Methods for Detecting an increased risk for coronary heart disease

ABSTRACT

The invention relates generally to an allele on human chromosome 9 associated with increased risk for coronary heart disease and the use or detection of such an allele in determining whether a human has an increased risk for coronary heart disease. In one aspect, the invention relates to methods for detecting a predisposition or propensity or susceptibility for coronary heart disease in a human, comprising detecting the presence of an allele on human chromosome 9 that is associated with an increased risk for coronary heart disease in a human. Disclosed are methods and compositions for determining whether a person carries an allele associated with increased risk for coronary atherosclerosis by determining whether the person has an RA-CHR9 allele, such as by determining whether the person has an RA-CHR9 allele-associated single nucleotide polymorphism (SNP). The invention also relates to kits for detecting the presence of an allele on chromosome 9 associated with an increased risk for coronary heart disease.

This application claims priority benefit to U.S. provisional patentapplication Ser. No. 60/927,361, filed May 2, 2007, which isincorporated herein by reference in its entirety.

1. INTRODUCTION

The invention relates generally to an allele on human chromosome 9associated with increased risk for coronary heart disease and the use ordetection of such an allele in determining whether a human has anincreased risk for coronary heart disease. In one aspect, the inventionrelates to methods for detecting a predisposition or propensity orsusceptibility for coronary heart disease in a human, comprisingdetecting the presence of an allele on human chromosome 9 that isassociated with an increased risk for coronary heart disease in a human.The invention also relates to kits for detecting the presence of anallele on chromosome 9 associated with an increased risk for coronaryheart disease.

2. BACKGROUND OF THE INVENTION

Coronary heart disease (CHD) is the single greatest cause of deathworldwide (Murray et at. (1997) Lancet 349: 1436; Mathers et al. (2006)PLoS Med, 3: e442). Coronary heart disease is a complex disease. Theidentification of genetic markers associated with increased risk forcoronary heart disease can be used to develop and implement strategiesto treat and to reduce the likelihood of a human developingcoronary-heart disease.

3. SUMMARY OF THE INVENTION

In one aspect, the invention provides methods and compositions fordetermining whether a person carries an allele associated with increasedrisk for coronary heart disease (e.g., coronary atherosclerosis ormyocardial infarction) by determining whether the person has an RA-CHR9allele. In certain embodiments, the RA-CHR9 allele is an approximately58 kb region extending from about position 22,062,301 to about position22,120,389 of human chromosome 9 that is significantly associated withcoronary heart disease. In a specific embodiment, the RA-CHR9 allele isa 58 kb region extending from 22,062,301 to 22,120,389 of humanchromosome 9 that is significantly associated with coronary heartdisease. In particular embodiments, the person has a family history ofcoronary atherosclerosis. In some embodiments, the person has a familyhistory of myocardial infarction.

In one embodiment, the invention provides a method for determiningwhether a person carries an allele associated with increased risk forcoronary heart disease, the method comprising using an in vitro assay todetect whether the person has an RA-CHR9 allele. In a specificembodiment, the invention provides a method for determining whether aperson carries an allele associated with increased risk for coronaryheart disease, the method comprising detecting one, two, three or more,all or any combination of the single nucleotide polymorphisms (“SNPs”)found in an RA-CHR9 allele, such as those SNPs in Table 7, Table 8,Table 10 or Table 11. Non-limiting examples of SNPs found in an RA-CHR9allele include those SNPs found at the following positions on humanchromosome 9: the SNP found at position 22062301; the SNP found atposition 22062638; the SNP found at position 22067543; the SNP found atposition 22062719; the SNP found at position 22071397; the SNP found atposition 22071850; the SNP found at position 22078090; the SNP found atposition 22078094; the SNP found at position 22078260; the SNP found atposition 22086055; the SNP found at position 22088574; the SNP found atposition 22088619; the SNP found at position 22090176; the SNP found atposition 22091702; the SNP found at position 22092165; the SNP found atposition 22093183; the SNP found at position 22093341; the SNP found atposition 22093813; the SNP found at position 22095927; the SNP found atposition 22096731; the SNP found at position 22100131; the SNP found atposition 22102241; the SNP found at position 22102427; the SNP found atposition 22104469; the SNP found at position 22104495; the SNP found atposition 22105026; the SNP found at position 22105286; the SNP found atposition 22106046; the SNP found at position 22106220; the SNP found atposition 22113766; the SNP found at position 22114123; the SNP found atposition 22114140; the SNP found at position 22115347; and the SNP foundat position 22115503. In a specific embodiment, the SNPs at rs10757274and rs2383206 are detected.

In another embodiment, the invention provides a method for determiningwhether a person carries an allele associated with increased risk forcoronary heart disease, the method comprising detecting one, two, threeor more, all or any combination of the insertions and/or deletions foundin an RA-CHR9 allele, such as those insertions and/or deletions in Table7 or 10. Non-limiting examples of insertions and deletions in an RA-CHR9allele include those insertions and deletions found at the followingpositions on human chromosome 9: the deletion found at position22078465; the insertion found at position 22089755; the insertion foundat position 22101587; and the insertion found at position 22110491.

In another embodiment, the invention provides a method for determiningwhether a person carries an allele associated with increased risk forcoronary heart disease, the method comprising detecting one, two, threeor more, all or any combination of the SNPs, insertions and/or deletionsfound in an RA-CHR9 allele, such as those insertions and/or deletions inTable 7, Table 8, Table 10 or Table 11. Non-limiting examples of SNPs,insertions and deletions found in an RA-CHR9 allele include those SNPs,insertions and/or deletions found at the following positions on humanchromosome 9: the SNP found at position 22062301; the SNP found atposition 22062638; the SNP found at position 22062719; the SNP found atposition 22071397; the SNP found at position 22071850; the SNP found atposition 22078090; the SNP found at position 22078094; the SNP found atposition 22078260; the deletion found at position 22078465; the SNPfound at position 22086055; the SNP found at position 22088574; the SNPfound at position 22088619; the insertion found at position 22089755;the SNP found at position 22090176; the SNP found at position 22091702;the SNP found at position 22092165; the SNP found at position 22093183;the SNP found at position 22093341; the SNP found at position 22093813;the SNP found at position 22095927; the SNP found at position 22096731;the SNP found at position 22100131; the insertion found at position22101587; the SNP found at position 22102241; the SNP found at position22102427; the SNP found at position 22104469; the SNP found at position22104495; the SNP found at position 22105026; the SNP found at position22105286; the SNP found at position 22106046; the SNP found at position22106220; the insertion found at position 22110491; the SNP found atposition 22113766; the SNP found at position 22114123; the SNP found atposition 22114140; the SNP found at position 22115347; and the SNP foundat position 22115503.

In another embodiment, the invention provides a method for determiningwhether a person carries an allele associated with increased risk forcoronary atherosclerosis, the method comprising using an in vitro assayto detect whether the person has an RA-CHR9 allele. In anotherembodiment, the invention provides a method for determining whether aperson carries an allele associated with increased risk for coronaryatherosclerosis, the method comprising the step of: determining whetherthe person has an RA-CHR9 allele. In certain embodiments, thedetermining step comprises detecting one, two, three or more or all ofthe single nucleotide polymorphisms (SNPs) found in an RA-CHR9 allele,such as those SNPs in Table 7, Table 8, Table 10 or Table 11. Inparticular embodiments, the determining step comprises detecting theallele using a method selected from the group consisting of: massspectroscopy, oligonucleotide microarray analysis, allele-specifichybridization, allele-specific PCR, and sequencing.

In another embodiment, the invention provides a method for determiningwhether a person carries an allele associated with increased risk ofhaving a myocardial infarction, the method comprising using an in vitroassay to detect whether the person has an RA-CHR9 allele. In a specificembodiment, the invention provides a method for determining whether aperson carries an allele associated with increased risk of having amyocardial infarction, the method comprising detecting in vitro one,two, three or more or all of the single nucleotide polymorphisms (SNPs)found in an RA-CHR9 allele, such as those SNPs in Table 7, Table 8,Table 10 or Table 11.

In another aspect, the invention provides a method for identifying ahuman subject at increased risk for coronary heart disease, comprisingusing an in vitro assay to detect the presence of a risk allele in anapproximately 58 kb region extending from approximately 22,062,301 toapproximately 22,120,389 of human chromosome 9 in a human subject thatis more frequently present in a population of humans with coronary heartdisease than in a population of humans that do not have coronary heartdisease, wherein the presence of the risk allele indicates that thehuman subject has an increased risk for coronary heart disease. Incertain embodiments, the risk allele is in a region extending fromposition 22,062,201+/−5808, 5000, 4000, 3000, 2500, 2000, 1500, 1101,1000, 550, 500, 450, 400, 350, 300, 250, 200, 150, 100 or 50 nucleotidesto position 22,120,389+/−5808, 5000, 4000, 3000, 2500, 2000, 1500, 1101,1000, 550, 500, 450, 400, 350, 300, 250, 200, 150, 100 or 50 nucleotidesof human chromosome 9. In a specific embodiment, the risk allele is in aregion extending from position 22,062,201 to position 22,120,389 ofhuman chromosome 9. In certain embodiments, the risk allele is at aposition on human chromosome 9 identified in Table 7, Table 8, or Table10 or Table 11. In one embodiment, the risk allele is at least one ofthe following positions on human chromosome 9: position 22062301;position 22062638; position 22067543; position 22062719; position22071397; position 22071850; position 22078090; position 22078094;position 22078260; position 22078465; position 22086055; position22088574; position 22088619; position 22089755; position 22090176;position 22091702; position 22092165; position 22093183; position22093341; position 22093813; position 22095927; position 22096731;position 22100131; position 22101587; position 22102241; position22102427; position 22104469; position 22104495; position 22105026;position 22105286; position 22106046; position 22106220; position2210491; position 22113766; position 22114123; position 22114140;position 22115347; and/or position 22115503. In another embodiment, therisk allele is at one or more, all or any combination of the following:rs9632884, rs6475606, rs10757272, rs10757274, rs4977574, rs2891168,rs1333042, rs2383206, rs1333048, or rs1333049. In a specific embodiment,the risk allele is at rs10757274 or rs2383206. In another specificembodiment, the risk alleles are rs10757274 and rs2383206.

In another aspect, the invention provides a method for identifying ahuman subject at increased risk for coronary heart disease, comprisingusing an in vitro assay to detect the presence of a human chromosome 9haplotype comprising a guanine nucleotide at position 22086055(rs10757274) and/or a guanine at position 22105026 (rs2383206) in ahuman subject, wherein the presence of the haplotype indicates that thehuman subject has an increased risk for coronary heart disease. In oneembodiment, the haplotype comprises a guanine nucleotide at position22086055 (rs10757274) and a guanine nucleotide at position 22105026(rs2383206) of chromosome 9. In another embodiment, the haplotypecomprises a guanine nucleotide at position 22086055 (rs10757274) and/ora guanine nucleotide at position 22105026 (rs2383206), and at least one,two, three, or more, all or any combination of the following SNPs: acytosine nucleotide at position 22062301 (rs9632884), a thyminenucleotide at position 22071850 (rs6475606), a thymine nucleotide atposition 22078260 (rs10757272), a guanine at position 22088574(rs4977574), a guanine nucleotide at position 22088619 (rs2891168), aguanine nucleotide at position 22093813 (rs1333042), a cytosinenucleotide at position 22115347 (rs1333048), and/or a cytosinenucleotide at position 22115503 (rs1333049). In another embodiment, thehaplotype comprises guanine nucleotide at position 22086055 (rs10757274)and/or a guanine nucleotide at position 22105026 (rs2383206), and atleast one, two, three or more, all or any combination of the following:a guanine nucleotide at position 22062264; a cytosine nucleotide atposition 22062301; an adenine nucleotide at position 22062638; a guaninenucleotide at position 22062719; a thymine nucleotide at position22071397; a thymine nucleotide at position 22071850; a thyminenucleotide at position 22078090; a guanine nucleotide at position22078094; a thymine nucleotide at position 22078260; a deletion atposition 22078465; a guanine nucleotide at position 2208874; a guaninenucleotide at position 22088619; an insertion at position 22089755; acytosine nucleotide position 22090176; a cytosine nucleotide at position22091702; a thymine nucleotide at position 22092165; a thyminenucleotide at position 22093183; a guanine nucleotide at position22093341; a guanine nucleotide at position 22093813; a cytosinenucleotide at position 22095927; an adenine nucleotide at position22096731; a cytosine nucleotide at position 22100131; an insertion atposition 22101587; a cytosine nucleotide at position 22102241; a guaninenucleotide at position 22102427; a cytosine nucleotide at position22104469; a guanine nucleotide at position 22104495; a cytosinenucleotide at position 22105286; a guanine nucleotide at position22106046; a cytosine nucleotide at position 22106220; an insertion atposition 22110491; a cytosine nucleotide at position 22113766; anadenine nucleotide at position 22114123; a thymine nucleotide atposition 22114140; a cytosine nucleotide at position 22115347; and/or acytosine at position 22115503. In a specific embodiment, the haplotypeis in a region extending from position 22062301 to 22120389 of humanchromosome 9.

In another aspect, the invention provides a method for identifying ahuman subject at increased risk for coronary heart disease, comprisingusing an in vitro assay to detect the presence of a polymorphism with alinkage disequilibrium of between 0.5 to 1, 0.5 to 0.90, or 0.5 to 0.80,or 0.5 to 0.75 with a risk allele in an approximately 58 kb regionextending from approximately 22,062,301 to approximately 22,120,389 ofhuman chromosome 9 in a human subject, wherein the presence of thepolymorphism indicates that the human subject has an increased risk forcoronary heart disease. In certain embodiments, the presence of apolymorphism with a linkage disequilibrium of at least r²=0.50, 0.55,0.60, 0.65, 0.70, 0.75, 0.80, or 0.85 with a risk allele in anapproximately 58 kb region extending from about 22,062,301 to about22,120,389 of human chromosome 9 in a human subject is detected. In someembodiments, a polymorphism with a linkage disequilibrium of at leastr²=0.89, 0.90, 0.91, 0.92, 0.93, 0.94, 0.95, 0.96, 0.97, 0.98 or 0.99with a risk allele in an approximately 58 kb region extending from about22,062,301 to about 22,120,389 of human chromosome 9 in a human subjectis detected. In a specific embodiment, a polymorphism with a linkagedisequilibrium of r²=0.89, 0.90, 0.91, 0.92, 0.93, 0.94, 0.95, 0.96,0.97, 0.98, 0.99 or 1 with a risk allele in an approximately 58 kbregion extending from about 22,062,301 to about 22,120,389 of humanchromosome 9 in a human subject is detected. In certain embodiments, therisk allele is in a region extending from position 22,062,301+/−5808,5000, 4000, 3000, 2500, 2000, 1500, 1101, 1000, 550, 500, 450, 400, 350,300, 250, 200, 150, 100 or 50 nucleotides to position 22,120,389+/−5808,5000, 4000, 3000, 2500, 2000, 1500, 1101, 1000, 550, 500, 450, 400, 350,300, 250, 200, 150, 100 or 50 nucleotides of human chromosome 9. In aspecific embodiment, the risk allele is in a region extending fromposition 22,062,301 to position 22,120,389 of human chromosome 9.

In another aspect, the invention provides a method for determiningwhether a person carries an allele associated with increased risk forcoronary heart disease e.g., coronary atherosclerosis or myocardialinfarction), comprising determining whether the person carries at leastone, two, three, four or more, all or any combination of the SNPs,insertions and/or deletions listed in Table 7, Table 8, Table 10 orTable 11, wherein the presence of one, two, three, four or more, all orany combination of the SNPs indicates that the person carries an alleleassociated with an increased risk for coronary heart disease. In oneembodiment, the invention provides a method for determining whether aperson carries an allele associated with increased risk for coronaryheart disease (e.g., coronary atherosclerosis or myocardial infarction),comprising determining whether the person carries at least one, two,three, four or more, all or any combination of the following SNPs,insertions and/or deletions on human chromosome 9: a guanine nucleotideat position 22062264; a cytosine nucleotide at position 22062301; anadenine nucleotide at position 22062638; a guanine nucleotide atposition 22062719; a thymine nucleotide at position 22071397; a thyminenucleotide position 22071850; a thymine nucleotide position 22078090; aguanine nucleotide at position 22078094; a thymine nucleotide position22078260; a deletion at position 22078465; a guanine nucleotide atposition 22088574; a guanine nucleotide at position 22088619; aninsertion at position 22089755; a cytosine nucleotide position 22090176;a cytosine nucleotide at position 22091702; a thymine nucleotide atposition 22092165; a thymine nucleotide at position 22093183; a guaninenucleotide at position 22093341; a guanine nucleotide at position22093813; a cytosine nucleotide at position 22095927; an adeninenucleotide at position 22096731; a cytosine nucleotide at position22100131; an insertion at position 22101587; a cytosine nucleotide atposition 22102241; a guanine nucleotide at position 22102427; a cytosinenucleotide at position 22104469; a guanine nucleotide at position22104495; a guanine nucleotide at 22105026; a cytosine nucleotide atposition 22105286; a guanine nucleotide at position 22106046; a cytosinenucleotide at position 22106220; an insertion at position 22110491; acytosine nucleotide at position 22113766; an adenine nucleotide atposition 22114123; a thymine nucleotide at position 22114140; a cytosinenucleotide at position 22115347; and/or a cytosine at position 22115503,wherein the presence of one, two, three, four or more, all or anycombination of the SNPs, insertions and/or deletions indicates that theperson carries an allele associated with an increased risk for coronaryheart disease. In a specific embodiment, the SNPs detected are atrs10757274 and rs2383206.

In another embodiment, the invention provides a method for determiningwhether a person carries an allele associated with increased risk forcoronary heart disease (e.g., coronary atherosclerosis or myocardialinfarction), comprising determining whether the person carries a guaninenucleotide at position 22086055 (rs10757274) and/or a guanine nucleotideat position 22105026 (rs2383206) on human chromosome 9, and at leastone, two, three, or more, all or any combination of the following SNPson human chromosome 9: a cytosine nucleotide at position 22062301(rs9632884), a thymine nucleotide at position 22071850 (rs6475606), athymine nucleotide at position 22078260 (rs10757272), a guanine atposition 22088574 (rs4977574), a guanine nucleotide at position 22088619(rs2891168), a guanine nucleotide at position 22093813 (rs1333042), acytosine nucleotide at position 22115347 (rs1333048), and/or a cytosinenucleotide at position 22115503 (rs1333049), wherein the presence ofone, two, three, or more, all or any combination of the SNPs indicatesthat the person carries an allele associated with an increased risk forcoronary heart disease. In a specific embodiment, the invention providesa method for determining whether a person carries an allele associatedwith increased risk for coronary heart disease (e.g., coronaryatherosclerosis or myocardial infarction), comprising determiningwhether the person carries a guanine nucleotide at position 22086055(rs10757274) and a guanine nucleotide at position 22105026 (rs2383206)on human chromosome 9, wherein the presence of the SNPs indicates thatthe person carries an allele associated with an increased risk forcoronary heart disease. In another embodiment, the invention provides amethod for determining whether a person carries an allele associatedwith an increased risk for coronary heart disease, comprisingdetermining whether the person carries a cytosine nucleotide at position22115503 (rs1333049) alone or in addition to one, two, three or moreSNPs, insertions and/or deletions found in the approximately 58 kbregion extending from about 2,062,301 to about 22,120,301 (in specificembodiments, in the region extending from position 22,062,301 toposition 22,120,389) of human chromosome 9, such as those describedherein (see, e.g., Table 7, Table 8, Table 10 and Table 11).

In another aspect, the invention provides a method for determiningwhether a human subject has an allele associated with an increased riskfor coronary heart disease, comprising using an in vitro assay to detectwhether a risk allele is present in the human subject, wherein the riskallele is in an approximately 58 kb region extending from approximately22,062,301 to approximately 22,120,389 of human chromosome 9 that ismore frequently present in a population of humans with coronary heartdisease than in a population of humans that do not have coronary heartdisease. In a specific embodiments, the risk allele is in a regionextending from 22,062,301 to 22,120,389 of human chromosome 9. Incertain embodiments, the risk allele is at a position on humanchromosome 9 identified in Table 7, Table 8, or Table 10 or Table 1. Inone embodiment, the risk allele is at least one of the followingpositions on human chromosome 9: position 22062301; position 22062638;position 22067543; position 22062719; position 22071397; position22071850; position 22078090; position 22078094; position 22078260;position 22078465; position 22086055; position 22088574; position22088619; position 22089755; position 22090176; position 22091702;position 22092165; position 22093183; position 22093341; position22093813; position 22095927; position 22096731; position 22100131;position 22101587; position 22102241; position 22102427; position22104469; position 22104495; position 22105026; position 22105286;position 22106046; position 22106220; position 22110491; position22113766; position 22114123; position 22114140; position 22115347;and/or position 2215503. In another embodiment, the risk allele is atone or more, all or any combination of the following: rs9632884,rs6475606, rs107572772, rs10757274, rs4977574, rs2891168, rs1333042,rs2383206, rs1333048, or rs1333049. In a specific embodiment, the riskallele is at rs10757274 or rs2383206.

In another aspect, the invention provides a method for determiningwhether a human subject has a haplotype associated with increased riskfor coronary heart disease, comprising using an in vitro assay to detectwhether a human chromosome 9 haplotype comprising a guanine nucleotideat position 22086055 (rs10757274) and/or a guanine at position 22105026(rs2383206) is present in a human subject. In one embodiment, thehaplotype comprises a guanine nucleotide at position 22086055(rs10757274) and a guanine nucleotide at position 22105026 (rs2383206)of chromosome 9. In another embodiment, the haplotype comprises aguanine nucleotide at position 22086055 (rs10757274) and/or a guaninenucleotide at position 22105026 (rs2383206), and at least one, two,three, or more, all or any combination of the following SNPs: a cytosinenucleotide at position 22062301 (rs9632884), a thymine nucleotide atposition 22071850 (rs6475606), a thymine nucleotide at position 22078260(rs10757272), a guanine at position 22088574 (rs4977574), a guaninenucleotide at position 22088619 (rs2891168), a guanine nucleotide atposition 22093813 (rs1333042), a cytosine nucleotide at position22115347 (rs1333048), and/or a cytosine nucleotide at position 22115503(rs1333049). In another embodiment, the haplotype comprises guaninenucleotide at position 22086055 (rs10757274) and/or a guanine nucleotideas position 22105026 (rs2383206), and at least one, two, three or more,all or any combination of the following: a guanine nucleotide atposition 22062264; a cytosine nucleotide at position 22062301; anadenine nucleotide at position 22062638; a guanine nucleotide atposition 22062719; a thymine nucleotide at position 22071397; a thyminenucleotide at position 22071850; a thymine nucleotide at position22078090; a guanine nucleotide at position 22078094; a thyminenucleotide position 22078260; a deletion at position 22078465; anucleotide guanine at position 2208874; a guanine nucleotide at position22088619; an insertion at position 22089755; a cytosine nucleotideposition 22090176; a cytosine nucleotide at position 22091702; a thyminenucleotide at position 22092165; a thymine nucleotide at position22093183; a guanine nucleotide at position 22093341; a guaninenucleotide at position 22093813; a cytosine nucleotide at position22095927; an adenine nucleotide at position 22096731; a cytosinenucleotide at position 22100131; an insertion at position 22101587; acytosine nucleotide at position 22102241; a guanine nucleotide atposition 22102427; a cytosine nucleotide at position 22104469; a guaninenucleotide at position 22104495; a cytosine nucleotide at position22105286; a guanine nucleotide at position 22106046; a cytosinenucleotide at position 22106220; an insertion at position 22110491; acytosine nucleotide at position 22113766; an adenine nucleotide atposition 22114123; a thymine nucleotide at position 22114140; a cytosinenucleotide at position 22115347; and/or a cytosine at position 22115503.In one embodiment, the haplotype is in a region extending from aboutposition 22062301 to about position 22120389 of human chromosome 9. Incertain embodiments, the haplotype is in a region extending fromposition 22062301+/−5808, 5000, 4000, 3000, 2500, 2000, 1500, 1101,1000, 550, 500, 250, 200, 150, 100 or 50 nucleotides to position2212039+/−5808, 5000, 4000, 3000, 2500, 2000, 1500, 1101, 1000, 550,500, 250, 200, 150, 100 or 50 nucleotides of human chromosome 9. In aspecific embodiment, the haplotype is in a region extending fromposition 22,062,301 to position 22,120,039 of human chromosome 9.

In another aspect, the invention provides a method for determining ahuman subject has a polymorphism associated with increased risk forcoronary heart disease, comprising using in vitro assay to detectwhether a polymorphism with a linkage disequilibrium of between 0.5 to1, 0.5 to 0.90, or 0.5 to 0.80, or 0.5 to 0.75 with a risk allele in anapproximately 58 kb region extending from approximately 22,062,301 toapproximately 22,120,389 of human chromosome 9 is present in a humansubject. In certain embodiments, the presence of a polymorphism with alinkage disequilibrium of at least r²=0.50, 0.55, 0.60, 0.65, 0.70,0.75, 0.80, or 0.85 with a risk allele in an approximately 58 kb regionextending from about 22,062,301 to about 22,120,389 of human chromosome9 in a human subject is detected. In some embodiments, a polymorphismwith a linkage disequilibrium of at least r²=0.89, 0.90, 0.91, 0.92,0.93, 0.94, 0.95, 0.96, 0.97, 0.98 or 0.99 with an approximately 58 kbrisk allele in a region extending from about 22,062,301 to about22,120,389 of human chromosome 9 in a human subject is detected. In aspecific embodiment, a polymorphism with a linkage disequilibrium ofr²=0.89, 0.90, 0.91, 0.92, 0.93, 0.94, 0.95, 0.96, 0.97, 0.98, 0.99 or 1with a risk allele in an approximately 58 kb region extending from about22,062,301 to about 22,120,389 of human chromosome 9 in a human subjectis detected. In some embodiments, the risk allele(s) is at rs10757274and rs2383206. In certain embodiments, the risk allele is in a regionextending from position 2206301+/−5808, 5000, 4000, 3000, 2500, 2000,1500, 1101, 1000, 550, 500, 250, 200, 150, 100, or 50 nucleotides toposition 22120389+/−5808, 5000, 4000, 3000, 2500, 2000, 1500, 1101,1000, 550, 500, 250, 200, 150, 100, or 50 nucleotides of humanchromosome 9. In a specific embodiment, the risk allele is in a regionextending from position 22,062,301 to position 22,120,039 of humanchromosome 9.

In addition to the SNPs and other polymorphisms demonstrated herein tocorrelate with CHD, such as listed in e.g., Table 7, Table 8 and Table10, in view of the teaching herein further SNPs and other polymorphismsas an indication of an increased risk of CHD that can routinely bedetected/identified. For example, additional SNPs present within theapproximately 58 kb region described herein (see, e.g., the SNPs listedin Table 11) can be identified as correlation with increased risk forCHD. Routine techniques, such as genotyping and other techniquesdescribed herein can be used to identify such SNPs and otherpolymorphisms.

In certain embodiments, the methods further comprise providing results,information, data, determinations and/or identifications obtained viathe methods described herein to an individual, clinic, or healthcareprofessional, e.g., a clinician, a medical doctor. In other embodiments,a person identified as having an increased risk for coronary heartdisease is administered a therapy to delay the onset, prevent the onsetor slow the progression of coronary heart disease. In some embodiments,a person identified as having an increased risk for a myocardialinfarction is administered a therapy to delay or prevent the onset of amyocardial infarction. In particular embodiments, a person characterizedfor increased risk for coronary atherosclerosis is treated with atherapy to delay the onset of or slow progression of the coronaryatherosclerosis, particularly wherein the therapy comprises alipid-lowering medication.

The invention provides reagents and kits for practicing the disclosedmethods. In one embodiment, the invention provides a kit comprising oneor more components for detecting the presence of an RA-CHR9 allele in ahuman subject.

3.1. Terminology

As used herein, reference to particular positions on human chromosome 9are defined based on NCBI Build 36 coordinates (see, e.g., NCBIAccession Number NC_(—)000009.10 and the following websites regardingthe NCBI Build 36 coordinates;http://dec2007.archive.ensembl.org/Homo_sapiens/assemblies.html; andhttp://www.ncbi.nlm.nih.gov/mapview/stats/BuildStats.cgi?taxid9606&build=36&ver=1)unless otherwise indicated.

As used herein, the terms “about” and “approximately”, unless otherwiseindicated, refer to a value that is no more than 10%, 8%, 5%, 2% or 1%above or below the value being modified by the term.

As used herein, the term “coronary heart disease” has its commonmeaning, for example, it refers to a disease caused by the narrowing ofthe small blood vessels that supply blood and oxygen to the heart andincludes symptoms and events resulting therefrom, such as (withoutlimitation) myocardial infarction, coronary atherosclerosis, angina, andcongestive heart failure. In certain embodiments, the term “coronaryheart disease” refers to myocardial infarction. In some embodiments, theterm “coronary heart disease” refers to coronary atherosclerosis. Incertain embodiments, the term “coronary heart disease” refers to theconditions of the humans enrolled in the Ottawa Heart Study, theAtherosclerosis Risk in Communities Study, The Copenhagen City HeartStudy and/or the Dallas Heart Study

As used herein, the term “elderly human” refers to a human 65 years orolder.

As used herein, the term “human adult” refers to a human that is 18years or older.

As used herein, the term “human child” refers to a human that is 1 yearto 18 years old.

As used herein, the term “human infant” refers to a newborn to 1 yearold year human.

As used herein, the term “human toddler” refers to a human that is 1year to 3 years old.

As used herein, the term “more frequently” in the context of an allele,haplotype or SNP means that a particular allele, haplotype or SNP ispresent at a statistically significantly higher frequency in apopulation of human subjects that have coronary heart disease relativeto a population of human subjects that do not have coronary heartdisease.

As used herein, the term “isolated” in the context of a nucleic acid(e.g., DNA, RNA, cDNA, etc.) refers to a nucleic acid that issubstantially free of cellular material or contaminating nucleic acidsfrom the cell or tissue source from which it is derived, orsubstantially free of chemical precursors or other chemicals whenchemically synthesized. The phrase “substantially free of cellularmaterial” includes preparations of a nucleic acid in which the nucleicacid is separated from cellular components of the cells from which it isisolated or produced. Thus, a nucleic acid that is substantially free ofcellular material includes preparations of a nucleic acid having lessthan about 30%, 25%, 20%, 15%, 10%, or 5% (by dry weight) of acontaminating nucleic acid (e.g., a heterologous nucleic acid). When thenucleic acid is produced by chemical synthesis, it is preferablysubstantially free of chemical precursors or other chemicals, i.e., itis separated from chemical precursors or other chemicals which areinvolved in the synthesis of the nucleic acid. Accordingly, suchpreparations of a nucleic acid have less than about 30%, 20%, 10%, 5%(by dry weight) of chemical precursors or compounds other than thenucleic acid of interest. In a specific embodiment, a nucleic aciddisclosed herein is isolated.

As used herein, the terms “person”, “patient” and “subject” are usedinterchangeably and refer to a human.

4. BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Study design for identification and validation of sequencevariants associated with coronary heart disease. Assuming independence,the probability of any single SNP achieving a nominal significance levelof 0.025 in all three studies with the associations being in the samedirection was 3.9×10−6 (0.0253×0.52), thus none of the 100,000 SNPswould be expected by chance to replicate consistently in all threecomparisons.

FIG. 2. Fine mapping of the genomic interval on chromosome 9 associatedwith coronary heart disease. A. SNPs spaced ˜5 kb apart in the intervalextending 175 kb upstream and downstream of rs10757274 and rs2383206were assayed in 500 cases and 500 controls from the Ottawa Heart Studypopulation. Bars represent P values (determined using Chi-Square tests)for differences in allele frequency between cases and controls.Arrowheads indicate rs10757274 and rs2383206. The asterisk representsrs518394. The risk interval is indicated with a gray box. The linkagedisequilibrium map indicates pairwise r2 values. Blocks are shaded on acontinuous scale where white represents an r2 of 0 and black representsan r2 of 1. B. Physical map of the region showing the location of therisk interval (gray box) relative to the noncoding RNA DQ485453 andadjacent genes: CDKN2A, ARF, and CDKN2B. Arrowheads indicate rs10757274and rs2383206 and the asterisk represents rs518394 (see Panel A).

5. DETAILED DESCRIPTION OF SPECIFIC EMBODIMENTS OF THE INVENTION

In one aspect, the invention provides a method for determining whether aperson carries an allele associated with increased risk for coronaryheart disease (“CHD”), the method comprising detecting whether theperson has an RA-CHR9 allele. In one embodiment, the invention providesa method for determining whether a person carries an allele associatedwith increased risk for coronary heart disease, the method comprisingdetecting one, two, three or more, all or any combination of the singlenucleotide polymorphisms (SNPs) found in an RA-CHR9 allele, such asthose SNPs in Table 7, Table 8, Table 10 or Table 11. In specificembodiments, a human subject is identified as being homozygous for aRA-CHR9 allele. As demonstrated in the working example below, a humansubject that is homozygous for a RA-CHR9 allele can exhibit a greaterlikelihood of developing coronary heart disease than a human subjectthat is heterozygous for the RA-CHR9 allele. In a specific embodiment, ahuman subject that is homozygous for a RA-CHR9 allele has at least a 5%,10%, 15% or 25% or a 5% to 25%, 5% to 30%, or 10% to 25% greaterlikelihood of developing coronary heart disease than a human subjectthat is heterozygous for the RA-CHR9 allele. In other embodiments, ahuman subject is identified as being heterozygous for a RA-CHR9 allele.In particular embodiments, the person subject to evaluation has a familyhistory of coronary atherosclerosis. In some embodiments, the personsubject to evaluation has a family history of myocardial infarction.

In one embodiment, the invention provides a method for determiningwhether a person carries an allele associated with increased risk forcoronary atherosclerosis, the method comprising detecting whether theperson has an RA-CHR9 allele. In one embodiment, the invention providesa method for determining whether a person carries an allele associatedwith increased risk for coronary atherosclerosis, the method comprisingthe step of: determining whether the person has an RA-CHR9 allele, asdetailed below. The RA-CHR9 allele may be detected by any suitable,specific technique known in the art, such as, for example, massspectroscopy, oligonucleotide microarray analysis, allele-specifichybridization, allele-specific PCR, and sequencing. In a specificembodiment, the determining step comprises determining whether theperson has an RA-CHR9 allele-associated single nucleotide polymorphism(SNP). Suitable SNPs are described below, and alternative suitableRA-CHR9 allele-associated SNPs are readily identified as describedbelow. In particular embodiments, the determining step comprisesdetermining whether the person has an RA-CHR9 allele-associated SNPselected from the group consisting of rs10757274 and rs2383206,rs6475606, rs1412832, rs10811645, and rs7865618.

In another embodiment, the invention provides a method for determiningwhether a human subject is at increased risk for coronary heart disease,comprising detecting whether a risk allele in an approximately 58 kbregion extending from approximately position 22,062,301 to approximatelyposition 22,120,389 of human chromosome 9 is present in a human subject,wherein the risk allele is more frequently present in a population ofhumans with coronary heart disease compared to a population of humansthat do not have coronary heart disease, and wherein the presence of therisk allele indicates that the human subject has an increased risk forcoronary heart disease. In some embodiments, the risk allele is in aregion extending from position 22,062,301+/−5808, 5000, 4407, 4000,3000, 2500, 2000, 1500, 1101, 1000, 550, 500, 250, 200, 150, 100 or 50nucleotides to position 22,120, 89+/−5808, 5000, 4407, 4000, 3000, 2500,2000, 1500, 1000, 500, 250, 200, 150, 100 or 50 nucleotides of humanchromosome 9. In a specific embodiment, the risk allele is in a regionextending from position 22,062,301 to position 22,120,389 of humanchromosome 9. In certain embodiments, the risk allele is at one or moreof the positions identified in Table 7, Table 8, Table 10 or Table 11.In specific embodiments, a human subject is identified as beinghomozygous for a risk allele. As demonstrated in the working examplebelow, a human subject that is homozygous for a risk allele can exhibita greater likelihood of developing coronary heart disease than a humansubject that is heterozygous for the risk allele. In a specificembodiment, a human subject that is homozygous for a risk allele is atleast a 5%, 10%, 15% or 25%, or a 5% to 25%, 5% to 30% or 10% to 25%more likely to develop coronary heart disease than a human subjectheterozygous for the risk allele. In other embodiments, a human subjectis identified as being heterozygous for a risk allele.

In another aspect, the invention provides a method for determiningwhether a human subject is at increased risk for coronary heart disease,comprising detecting whether a human chromosome 9 haplotype comprising aguanine nucleotide at position 22086055 (rs10757274) and/or a guaninenucleotide at position 22105026 (rs2383206) is present in a humansubject, wherein the presence of the haplotype indicates that the humansubject has an increased risk for coronary heart disease. In specificembodiments, a human subject is identified as being homozygous for ahaplotype. As demonstrated in the working example below, a human subjectthat is homozygous for a chromosome 9 haplotype can exhibit a greaterlikelihood of developing coronary heart disease than a human subjectthat is heterozygous for the haplotype. In a specific embodiment, ahuman subject that is homozygous for a chromosome 9 haplotype has atleast a 5%, 10%, 15% or 25%, or a 5% to 25%, 5% to 30% or 10% to 25%greater likelihood of developing coronary heart disease than a humansubject that is heterozygous for the haplotype. In other embodiments, ahuman subject is identified as being heterozygous for a chromosome 9haplotype allele. In one embodiment, the haplotype comprises a guaninenucleotide at positions 22086055 (rs10757274) and 22105026 (rs2383206)of chromosome 9. In another embodiment, the haplotype comprises aguanine nucleotide at position 22078260 (rs10757274) and/or a guaninenucleotide at position 22105026 (rs2383206), and at least one, two,three, or more, all or any combination of the SNPs recited in Table 7,Table 8, Table 10 or Table 11. In a specific embodiment, the haplotypeis in an approximately 58 kb region extending from about position22062301 to about 22120389 of human chromosome 9. In certainembodiments, the haplotype is in a region extending from position22,062,301+/−5808, 5000, 4407, 4000, 3000, 2500, 2000, 1500, 1101, 1000,550, 500, 250, 200, 150, 100 or 50 nucleotides to position22,120.389+/−5808, 4407, 4000, 3000, 2000, 1500, 1100, 1000, 550, 500,250, 200, 150, 100 or 50 nucleotides of human chromosome 9.

In another aspect, the invention provides a method for determiningwhether a human subject is at increased risk for coronary heart disease,comprising detecting whether a polymorphism with a linkagedisequilibrium of between 0.5 to 1, 0.5 to 0.90, or 0.5 to 0.80, or 0.5to 0.75 with a risk allele in an approximately 58 kb region extendingfrom about 22,062,301 to about 22,120,389 of human chromosome 9 ispresent in a human subject, wherein the presence of the polymorphismindicates that the human subject has an increased risk for coronaryheart disease. In some embodiments, the haplotype is in a regionextending from position 22,062,301+/−5808, 5000, 4407, 4000, 3000, 2500,2000, 1500, 1101, 1000, 550, 500, 250, 200, 150, 100 or 50 nucleotidesto position 22,120,389+/−5808, 4407, 4000, 3000, 2000, 1500, 1100, 1000,550, 500, 250, 200, 150, 100 or 50 nucleotides of human chromosome 9. Ina specific embodiment, the risk allele is in a region extending fromposition 22,062,301 to 22,120,389 of human chromosome 9. In specificembodiments, a human subject is identified as being homozygous for apolymorphism. As demonstrated in the working example below, a humansubject that is homozygous for a polymorphism can exhibit a greaterlikelihood of developing coronary heart disease than a human subjectthat is heterozygous for the polymorphism. In a specific embodiment, ahuman subject that is homozygous for the haplotype has at least a 5%,10%, 15% or 25% or a 5% to 25%, 5% to 30%, or 10% to 25% greaterlikelihood of developing coronary heart disease that a human subjectthat is heterozygous for the haplotype. In other embodiments, a humansubject is identified as being heterozygous for a polymorphism.

In another aspect, the invention provides a method for determiningwhether a person carries an allele associated with increased risk forcoronary heart disease (e.g., coronary atherosclerosis or myocardialinfarction), comprising detecting whether a person carries at least one,two, three, four or more, all or any combination of the following SNPs,insertions, and/or deletions listed in Table 7, Table 8, Table 10 and/orTable 11, wherein the presence of at least one, two, three, four ormore, all or any combination of the SNPs indicates that the humansubject is at increased risk for coronary heart disease. In specificembodiments, a human subject is identified as being homozygous for aSNP(s). As demonstrated in the working example below, a human subjectthat is homozygous for a SNP(s) can exhibit a greater likelihood (e.g.,at least a 5%, 10%, 15% or 25% or a 5% to 25%, 5% to 30%, or 10% to 25%higher likelihood) of developing coronary heart disease than a humansubject that is heterozygous for the SNP(s). In other embodiments, ahuman subject is identified as being heterozygous for a SNP(s).

In certain embodiments, embodiments, the methods further compriseproviding results, information, data, determinations and/oridentifications obtained via the methods described herein to anindividual, clinic, or healthcare professional, e.g., a clinician, amedical doctor. In other embodiments, a person identified as having anincreased risk for coronary heart disease is administered a therapy todelay the onset, prevent the onset or slow the progression of coronaryheart disease. In some embodiments, a person identified as having anincreased risk for a myocardial infarction is administered a therapy todelay or prevent the onset of a myocardial infarction. In particularembodiments, a person characterized for increased risk for coronaryatherosclerosis is treated with a therapy to delay onset of or slowprogression of the coronary atherosclerosis, particularly wherein thetherapy comprises a lipid-lowering medication.

The invention further provides reagents and kits for practicing thedisclosed methods.

5.1. Methods for Detecting Increased Risk of Coronary Heart Disease

In one aspect, the invention provides a method for determining whether ahuman subject has an allele associated with increased-risk for coronaryheart disease (e.g., coronary atherosclerosis or myocardial infarction),the method comprising detecting whether RA-CHR9 allele is present in ahuman subject. In some embodiments, the human subject is identified ashaving or determined to have an increased risk for coronary heartdisease if the RA-CHR9 allele is detected. In some embodiments, theRA-CHR9 allele is approximately 58 kb and extends from about 22,062,301to about 22,120,389 of human chromosome 9. In certain embodiments, theRA-CHR9 allele extends from position 22,062,301+/−5508, 4407, 4000,3000, 2500, 2000, 1500, 1101, 1000, 550, 500, 250, 200, 150, 100 or 50nucleotides to position 22,120,389+/−5508, 4407, 4000, 3000, 2000, 1500,1100, 1000, 550, 500, 250, 200, 150, 100 or 50 nucleotides of humanchromosome 9. In some embodiments, the RA-CHR9 allele extends fromposition 22,062,040 to position 22,120,389 of human chromosome 9. Insome embodiments, the RA-CHR9 allele extends from position 22,062,040 toposition 22,120,389 of human chromosome 9. In some embodiments, thehuman subject is homozygous for the RA-CHR9 allele. In otherembodiments, the human subject is heterozygous for the RA-CHR9 allele.The presence of an RA-CHR9 allele in a human subject can be detected inan in vitro assay, e.g., a nucleic acid sample (see Section 5.3 below)and techniques known to one of skill in the art or described in Section5.4 below.

In certain embodiments, in addition to detecting the presence of anRA-CHR9 allele, one or more polymorphisms identified as being associatedwith an increased likelihood of coronary heart disease, such as thosedescribed in International Publication Nos. WO 2007/006862, WO2004/03576, WO 2004/035741, and WO 2006/105439; Shiffman et al (2005) AmJ Hum Genet 77: 596-605; Shiffman et al (2006) Arteriosclerosis,Thrombosis, and Vascular Biology 26: 1613-1618; Iakoubova et al. (2006)Arteriosclerosis, Thrombosis, and Vascular Biology 26: 2763-2768;Morrison et al. (2007) American Journal of Epidemiology 166: 28-35; Lukeet al. (2007) Arteriosclerosis, Thrombosis, and Vascular Biology 27:2030-2036; Shiffman et al. (2008) Arteriosclerosis, Thrombosis, andVascular Biology 28: 173-179; Iakoubova et al. (2008) J Am Coll Cardiol51: 449-455; Iakoubova et al. (2008) J Am Coll Cardiol 51: 435-443;Shiffman et al. (2008) J Am Coll Cardiol 51:444-448; Bare et al. (2007)Genetics in Medicine 9: 682-689; Helgadottir et al. (2004) Nat. Genet.36: 233-239; and U.S. Patent Application Publication Nos. 2007/0280917,2006/0019269, 2005/0282855, 2005/0164220, 2005/0113408, 2005/0112611,2007/0031847, 2006/0228715, 2007/0072821, 2006/0223093, and 2005/0272054(each of which are incorporated herein by reference) is also detected.

In one embodiment, the invention provides a method for determiningwhether a human subject has an allele associated with increased risk forcoronary heart disease (e.g., coronary atherosclerosis or myocardialinfarction), the method comprising detecting whether one, two, three ormore, all or any combination of the SNPs, insertions and/or deletionsfound in an RA-CHR9 allele, such as those SNPs, insertions and/ordeletions in Table 7, Table 8, Table 10 or Table 11, are present,wherein the detection of such SNPs insertions and/or deletions indicatesthat the human subject has an allele associated with coronary heartdisease. In some embodiments, the human subject is identified as havingor determined to have an increased risk for coronary heart disease ifsuch an allele is detected. The presence of said one, two, three or moreor all of the SNPs, insertions and/or deletions found in an RA-CHR9allele in a human subject can be detected in an in vitro assay, e.g., anucleic acid sample (see Section 5.3 below) and techniques known to oneof skill in the art or described in Section 5.4 below. Non-limitingexamples of SNPs, insertions, and deletions found in an RA-CHR9 alleleinclude: the SNP found at position 22062264; the SNP found at position22062301; the SNP found at position 22062638; the SNP at position22067543; the SNP found at position 22062719; the SNP found at position22071397; the SNP found at position 22071850; the SNP found at position22078090; the SNP found at position 22078094; the SNP found at position22078260; the deletion found at position 22078465; the SNP found atposition 22086055; the SNP found at position 22088574; the SNP found atposition 22088619; the insertion found at position 22089755; the SNPfound at position 22090176; the SNP found at position 22091702; the SNPfound at position 22092165; the SNP found at position 22093183; the SNPfound at position 22093341; the SNP found at position 22093813; the SNPfound at position 22095927; the SNP found at position 22096731; the SNPfound at position 22100131; the insertion found at position 22101587;the SNP found at position 22102241; the SNP found at position 22102427;the SNP found at position 22104469; the SNP found at position 22104495;the SNP found at position 22105026; the SNP found at position 22105286;the SNP found at position 22106046; the SNP found at position 22106220;the insertion found at position 22110491; the SNP found at position22113766; the SNP found at position 22114123; the SNP found at position22114140; the SNP found at position 22115347; and the SNP found atposition 22115503. In some embodiments, the human subject is homozygousfor a SNP. In other embodiments, the human subject is heterozygous for aSNP.

In another aspect, the invention provides a method for determiningwhether a human subject carries an allele associated with an increasedrisk for coronary heart disease (e.g., coronary atherosclerosis ormyocardial infarction), comprising detecting whether one, two, three,four or more risk alleles in an approximately 58 kb region extendingfrom about 22,062,301 to about 22,120,389 of human chromosome 9 that aremore frequently present in a population of humans with coronary heartdisease compared to a population of humans that do not have coronaryheart disease are present in a human subject. In some embodiments, thehuman subject is identified as having or determined to have an increasedrisk for coronary heart disease if such an allele is detected. In someembodiments, the risk allele is in an approximately 58 kb regionextending from about position 22,062,301 to about position 22,120,389 ofhuman chromosome 9. In some embodiments, the risk allele is in a regionextending from position 22,062,301+/−5808, 5000, 4407, 4000, 3000, 2500,2000, 1500, 1101, 1000, 550, 500, 250, 200, 150, 100 or 50 nucleotidesto position 22,120,389+/−5808, 5000, 4407, 4000, 3000, 2500, 2000, 1500,1101, 1000, 550, 500, 250, 200, 150, 100 or 50 nucleotides of humanchromosome 9. In a specific embodiment, the risk allele is in a regionextending from position 22,062,301 to 22,120,389 of human chromosome 9.In some embodiments, the human subject is homozygous for the riskallele. In other embodiments, the human subject is heterozygous for therisk allele. The presence of one, two, three, four or more of the riskalleles can be detected using an in vitro assay, e.g., a nucleic acidsample (see Section 5.3 below) and techniques known to one of skill inthe art or described in Section 5.4 below.

In certain embodiments, one, two, three, four or more of the riskalleles is/ere at one, two, three, four or more positions identified inTable 7, Table 8, Table 10 or Table 11. In one embodiment, one, two,three, four or more of the risk alleles is at one, two, three, four ormore of the following positions of human chromosome 9: position22062264; position 22062301; position 22062638; position 22067543;position 22062719; position 22071397; position 22071850; position22078090; position 22078094; position 22078260; position 22086055;position 22088574; position 22088619; position 22078465; position22089755; position 22090176; position 22091702; position 22092165;position 22093183; position 22093341; position 22093813; position22095927; position 22096731; position 22100131; position 22101587;position 22102241; position 22102427; position 22104469; position22104495; position 22105026; position 22105286; position 22106046;position 22106220; position 22110491; position 22113766; position22114123; position 22114140; position 22115347; and/or position22115503. In another embodiment, one, two, three, four or more of therisk alleles is at one, two, three, four or more of the following:rs9632884, rs6475606, rs10757272, rs10757274, rs4977574, rs2891168,rs1333042, rs2383206, rs1333048, or rs1333049. In a specific embodiment,at least one or two of the risk alleles is at rs10757274 and/orrs2383206.

In certain embodiments, in addition to detecting risk alleles in anapproximately 58 kb region extending from about 22,062,301 to about22,120,389 of chromosome 9, one or more polymorphisms identified asbeing associated with an increased likelihood of coronary heart disease,such as those described in International Publication Nos. WO2007/006862, WO 2004/03576, WO 2004/035741, and WO 2006/105439; Shiffmanet al (2005) Am J Hum Genet 77: 596-605; Shiffman et at (2006)Arteriosclerosis, Thrombosis, and Vascular Biology 26: 1613-1618;Iakoubova et al. (2006) Arteriosclerosis, Thrombosis, and VascularBiology 26: 2763-2768; Morrison et al. (2007) American Journal ofEpidemiology 166: 28-35; Luke et al. (2007) Arteriosclerosis,Thrombosis, and Vascular Biology 27: 2030-2036; Shiffman et al. (2008)Arteriosclerosis, Thrombosis, and Vascular Biology 28: 173-179;Iakoubova et al. (2008) J Am Coll Cardiol 51: 449-455; Iakoubova et al.(2008) J Am Coll Cardiol 51: 435-443; Shiffman et al. (2008) J Am CollCardiol 51:444-448; Bare et al. (2007) Genetics in Medicine 9: 682-689;Helgadottir et al. (2004) Nat. Genet. 36: 233-239; and U.S. PatentApplication Publication Nos. 2007/0280917, 2006/0019269, 2005/0282855,2005/0164220, 2005/0113408, 2005/0112611, 2007/0031847, 2006/0228715,2007/0072821, 2006/0223093, and 2005/0272054 (each of which areincorporated by herein by reference) is also detected. In a specificembodiment, the presence of one, two or more risk alleles indicates thatthe human subject has a 10% to 25%, 10% to 20%, 10% to 15%, 12.5% to15%, 10% to 30%, 10% to 40% or 10% to 50% increased risk for coronaryheart disease.

In another aspect, the invention provides a method for determiningwhether a human subject carries a haplotype associated with increasedrisk for coronary heart disease (e.g., coronary atherosclerosis ormyocardial infarction), comprising detecting whether a human chromosome9 haplotype comprising a guanine nucleotide at position 220086055(rs10757274) and/or a guanine nucleotide at position 22105026(rs2383206) is present in a human subject. In some embodiments, thehuman subject is identified as having or determined to have an increasedrisk for coronary heart disease if the haplotype is detected. In someembodiments, the human subject is homozygous for the haplotype. In otherembodiments, the human subject is heterozygous for the haplotype. Thepresence of the haplotype in a human subject can be detected using an invitro assay, e.g., a nucleic acid sample (see Section 5.3 below) andtechniques known to one of skill in the art or described in Section 5.4below. In a specific embodiment, the presence of the haplotype indicatesthat the human subject has a 10% to 25%, 10% to 20%, 10% to 15%, 12.5%to 15%, 10% to 30%, 10% to 40% or 10% to 50% increased risk for coronaryheart disease. In one embodiment, the haplotype comprises a guaninenucleotide at positions 22086055 (rs10757274) and 22105026 (rs2383206)of human chromosome 9. In another embodiment, the haplotype comprises aguanine nucleotide at position 220086055 (rs10757274) and/or a guaninenucleotide at position 22105026 (rs2383206) on human chromosome 9, andat least one, two, three, or more, all or any combination of thefollowing SNPs on human chromosome 9: a cytosine nucleotide at position22062301 (rs9632884), a thymine nucleotide at position 22071850(rs6475606), a thymine nucleotide at position 22078260 (rs10757272), aguanine at position 22088574 (rs4977574), a guanine nucleotide atposition 22088619 (rs2891168), a guanine nucleotide at position 22093813(rs1333042), a cytosine nucleotide at position 22115347 (rs1333048),and/or a cytosine nucleotide at position 22115503 (rs1333049). Inanother embodiment, the haplotype comprises a guanine nucleotide atposition 22086055 (rs1075724) and/or a guanine nucleotide at position22105026 (rs283206) and at least one, two, three or more or all of thefollowing on human chromosome 9: a guanine nucleotide at position22062264; a cytosine nucleotide at position 22062301; an adeninenucleotide at position 22062638; a guanine nucleotide at position22062719; a thymine nucleotide at position 22071397; a thyminenucleotide position 22071850; an thymine nucleotide position 22078090; aguanine nucleotide at position 22078094; a thymine nucleotide position22078260; a deletion at position 22078465; a guanine nucleotide atposition 22088574; a guanine nucleotide at position 22088619; aninsertion at position 22089755; a cytosine nucleotide position 22090176;a cytosine nucleotide at position 22091702; a thymine nucleotide atposition 22092165; a thymine nucleotide at position 22093183; a guaninenucleotide at position 22093341; a guanine nucleotide at position22093813; a cytosine nucleotide at position 22095927; an adeninenucleotide at position 22096731; a cytosine nucleotide at position22100131; an insertion at position 22101587; a cytosine nucleotide atposition 22102241; a guanine nucleotide at position 22102427; a cytosinenucleotide at position 22104469; a guanine nucleotide at position22104495; a cytosine nucleotide at position 22105286; a guaninenucleotide at position 22106046; a cytosine nucleotide at position22106220; an insertion at position 22110491; a cytosine nucleotide atposition 22113766; an adenine nucleotide at position 22114123; a thyminenucleotide at position 22114140; a cytosine nucleotide at position22115347; and/or a cytosine at position 22115503. In some embodiments,the haplotype is in an approximately 58 kb region extending from aboutposition 22,062,301 to about 22,120,389 of human chromosome 9. Incertain embodiments, the haplotype is in a region extending fromposition 22,062,301+/−5808, 5000, 4407, 4000, 3000, 2500, 2000, 1500,1101, 1000, 550, 500, 250, 200, 150, 100 or 50 nucleotides to position22,120,398+/−5808, 5000, 4407, 4000, 3000, 2500, 2000, 1500, 1101, 1000,550, 500, 250, 200, 150, 100 or 50 nucleotides of human chromosome 9. Ina specific embodiment, the haplotype is in a region extending fromposition 22062301 to 22120389 of human chromosome 9.

In certain embodiments, in addition to detecting a human chromosome 9haplotype comprising a guanine nucleotide at position 22086055(rs10757274) and/or 22105026 (rs2383206), one or more polymorphismsidentified as being associated with an increased likelihood of coronaryheart disease, such as those described in International Publication No.WO 2007/006862, WO 2004/03576, WO 2004/035741, and WO 2006/105439;Shiffman et al (2005) Am J Hum Genet 77: 596-605; Shiffman et al (2006)Arteriosclerosis, Thrombosis, and Vascular Biology 26: 1613-1618;Iakoubova et al. (2006) Arteriosclerosis, Thrombosis, and VascularBiology 26: 2763-2768; Morrison et al. (2007) American Journal ofEpidemiology 166: 28-35; Luke et al. (2007) Arteriosclerosis,Thrombosis, and Vascular Biology 27: 2030-2036; Shiffman et al, (2008)Arteriosclerosis, Thrombosis, and Vascular Biology 28: 173-179;Iakoubova et al. (2008) J Am Coll Cardiol 51: 449-455; Iakoubova et al.(2008) J Am Coll Cardiol 51: 435-443; Shiffman et al. (2008) J Am CollCardiol 51:444-448; Bare et al. (2007) Genetics in Medicine 9: 682-689;Helgadottir et al. (2004) Nat. Genet. 36: 233-239; and U.S. PatentApplication Publication Nos. 2007/0280917, 2006/0019269, 2005/0282855,2005/0164220, 2005/0113408, 2005/0112611, 2007/0031847, 2006/0228715,2007/0072821, 2006/0223093, and 2005/0272054 (each of which areincorporated by herein by reference) is also detected.

In another aspect, the invention provides a method for determiningwhether a human subject has allele associated with increased risk forcoronary heart disease (e.g., coronary atherosclerosis or myocardialinfarction), comprising detecting whether a polymorphism with a linkagedisequilibrium of between 0.5 to 1, 0.5 to 0.90, 0.5 to 0.80, 0.5 to0.75, 0.75 to 1, or 0.85 to 1 with a risk allele in an approximately 58kb region extending from about 22,062,301 to about 22,120,389 ofchromosome 9 is present in a human subject. In some embodiments, thehuman subject is determined to have or identified as having an increasedrisk for coronary heart disease if the polymorphism is detected. In someembodiments, the human subject is homozygous for the polymorphism. Inother embodiments, the human subject is heterozygous for thepolymorphism. In certain embodiments, the presence of a polymorphismwith a linkage disequilibrium of at least r²=0.50, 0.55, 0.60, 0.65,0.70, 0.75, 0.80 or 0.85 with a risk allele in an approximately 58 kbregion extending from about 22,062,301 to about 22,120,389 of humanchromosome 9 in a human subject is detected. In some embodiments, apolymorphism with a linkage disequilibrium of at least r²=0.89, 0.90,0.91, 0.92, 0.93, 0.94 0.95, 0.96, 0.97, 0.98 or 0.99 with a risk allelein an approximately 58 kb region extending from about 22,062,301 toabout 22,120,389 of human chromosome 9 in a human subject is detected.In a specific embodiment, a polymorphism with a linkage disequilibriumof r²=0.89, 0.90, 0.91, 0.92, 0.93, 0.94 0.95, 0.96, 0.97, 0.98, 0.99 or1 with a risk allele in an approximately 58 kb region extending fromabout 22,062,301 to about 22,120,389 of human chromosome 9 in a humansubject is detected. In some embodiments, the risk allele is in a regionextending from position 22,062,301+/−5808, 5000, 4407, 4000, 3000, 2500,2000, 1500, 1101, 1000, 550, 500, 250, 200, 150, 100 or 50 nucleotidesto position 22,120,3889+/−5808, 5000, 4407, 4000, 3000, 2500, 2000,1500, 1101, 1000, 550, 500, 250, 200, 150, 100 or 50 nucleotides ofhuman chromosome 9. In a specific embodiment, the risk allele is in aregion extending from position 22,062,301 to position 22,120,389 ofhuman chromosome 9.

In one embodiment, the risk allele is a SNP, insertion or deletionlisted in Table 7, Table 8, Table 10 or Table 11. In one embodiment, therisk allele is at rs10757274 or rs2383206. In another embodiment, therisk allele is at rs9632884, rs6475606, rs10757272, rs10757274,rs4977574, rs2891168, rs1333042, rs2383206, rs1333048 or rs1333049. Thepresence of the polymorphism in the human subject can be detected usingan in vitro assay, e.g., a nucleic acid sample (see Section 5.3 below)and techniques known to one of skill in the art or described in Section5.4 below. In a specific embodiment, the presence of the polymorphismindicates that the human subject has a 10% to 25%, 10% to 20%, 10% to15%, 12.5% to 15%, 10% to 30%, 10% to 40% or 10% to 50% increased riskfor coronary heart disease.

In certain embodiments, in addition to detecting a polymorphism in anapproximately 58 kb region extending from about 22,062,301 to about22,120,389 of human chromosome 9 with a linkage disequilibrium of atleast r²=0.5, 0.55, 0.65, 0.70, 0.75, 0.85, 0.89, 0.90, 0.95 or 1, oneor more polymorphisms identified as being associated with an increasedlikelihood of coronary heart disease, such as those described inInternational Publication No. WO 2007/006862, WO 2004/03576. WO2004/035741, and WO 2006/105439; Shiffman et al (2005) Am J Hum Genet77: 596-605; Shiffman et al (2006) Arteriosclerosis, Thrombosis, andVascular Biology 26: 1613-1618; Iakoubova et al. (2006)Arteriosclerosis, Thrombosis, and Vascular Biology 26: 2763-2768;Morrison et al. (2007) American Journal of Epidemiology 166: 28-35; Lukeet al. (2007) Arteriosclerosis, Thrombosis, and Vascular Biology 27:2030-2036; Shiffman et al. (2008) Arteriosclerosis, Thrombosis, andVascular Biology 28: 173-179; Iakoubova et al. (2008) J Am Coll Cardiol51: 449-455; Iakoubova et al. (2008) J Am Coll Cardiol 51: 435-443;Shiffman et al. (2008) J Am Coll Cardiol 51:444-448; Bare t al. (2007)Genetics in Medicine 9: 682-689; Helgadottir et al. (2004) Nat. Genet.36: 233-239; and U.S. Patent Application Publication Nos. 2007/0280917,2006/0019269, 2005/0282855, 2005/0164220, 2005/013408, 200510112611,2007/0031847, 2006/0228715, 2007/0072821, 2006/0223093, and 2005/0272054(each of which are incorporated by herein by reference) is alsodetected.

Techniques for determining whether a polymorphism is in linkagedisequilibrium with a risk allele are well-known to the one of skill inthe art. See e.g., the following for information concerning linkagedisequilibrium in the human genome: Wall et al., “Haplotype blocks andlinkage disequilibrium in the human genome”, Nat Rev Genet. 2003 August;4(8):587-97; Garner et al., “On selecting markers for associationstudies: patterns of linkage disequilibrium between two and threediallelic loci”, Genet Epidemiol. 2003 January; 24(1):57-67; Ardlie etal., “Patterns of linkage disequilibrium in the human genome”, Nat RevGenet. 2002 April; 3(4):299.309 (erratum in Nat Rev Genet 2002 July;3(7):566); and Remm et al., “High-density genotyping and linkagedisequilibrium in the human genome using chromosome 22 as a model”; CurrOpin Chem Biol. 2002 February; 6(1):24-30; Haldane J B S (1919) Thecombination of linkage values, and the calculation of distances betweenthe loci of linked factors. J Genet 8:299-309; Mendel, G. (1866)Versuche uber Pflanzen-Hybriden. Verhandlungen des naturforschendenVereines in Brunn [Proceedings of the Natural History Society of Brunn];Lewin 8 (1990) Genes IV Oxford University Press, New York, USA; Hartl DL and Clark A G (1989) Principles of Population Genetics 2.sup.nd ed.Sinauer Associates, Inc. Sunderland, Mass., USA; Gillespie J H (2004)Population Genetics: A Concise Guide. 2.sup.nd ed. Johns HopkinsUniversity Press. USA; Lewontin R C (1964) The interaction of selectionand linkage. 1. General considerations; heterotic models. Genetics49:49-67; Hoel P G (1954) Introduction to Mathematical Statistics2.sup.nd ed. John Wiley & Sons, Inc. New York, USA; Hudson R R (2001)Two-locus sampling distributions and their application. Genetics159:1805-1817; Dempster A P, Laird N M, Rubin D B (1977) Maximumlikelihood from incomplete data via the EM algorithm. J R Stat Soc39:1-38; Excoffier L, Slatkin M (1995) Maximum-likelihood estimation ofmolecular haplotype frequencies in a diploid population. Mol Biol Evol12(5):921-927; Tregouet D A, Escolano S, Tiret L, Mallet A, Golmard J L(2004) A new algorithm for haplotype-based association analysis: theStochastic-EM algorithm. Ann Hum Genet 68(Pt 2):165-177; Long A D andLangley C H (1999) The power of association studies to detect thecontribution of candidate genetic loci to variation in complex traits.Genome Research 9:720-731; Agresti A (1990) Categorical Data Analysis.John Wiley & Sons, Inc. New York, USA; Lange K (1997) Mathematical andStatistical Methods for Genetic Analysis. Springer-Verlag New York, inc.New York, USA; The International HapMap Consortium (2003) TheInternational HapMap Project. Nature 426:789-796; The InternationalHapMap Consortium (2005) A haplotype map of the human genome. Nature437:1299-1320; Thorisson G A, Smith A V Krishnan L, Stein L D (2005),The International HapMap Project Web Site, Genome Research 15:1591-1593;McVean G, Spencer C C A, Chaix R (2005) Perspectives on human geneticvariation from the HapMap project. PLoS Genetics 1(4):413-418;Hirschhorn J N, Daly M J (2005) Genome-wide association studies forcommon diseases and complex traits. Nat Genet 6:95-108; Schrodi S J(2005) A probabilistic approach to large-scale association scans: asemi-Bayesian method to detect disease-predisposing alleles. SAGMB4(1):3 1; Wang W Y S, Barratt B J, Clayton D G, Todd J A (2005)Genome-wide association studies: theoretical and practical concerns. NatRev Genet 6:109-118. Pritchard J K, Przeworski M (2001) Linkagedisequilibrium in humans: models and data. Am J Hum Genet 69:1-14. Theparameter r² is commonly used in the genetics art to characterize theextent of linkage disequilibrium between two genetic loci (see, e.g.,Hudson et al. (2001) Genetics 159:1805-1817).

In another aspect, the invention provides a method for determiningwhether a human subject carries a SNP, insertion and/or deletionassociated with an increased risk for coronary heart disease (e.g.,coronary atherosclerosis or myocardial infarction), comprising detectingthe whether at least one, two, three, four or more or all of the SNPs,insertions and/or deletions listed in Table 7, Table 8, Table 10 orTable 11 is present in the human subject. In some embodiments, the humansubject is identified as having or determined to have an increased riskfor developing coronary heart disease if the SNP(s), insertion(s) and/ordeletion(s) is/arc detected. In a specific embodiment, the presence ofone, two, three four or more or all SNPs, insertions and/or deletionslisted in Table 7, Table 8, Table 10 or Table 11 indicates that thehuman subject has a 10% to 25%, 10% to 20%, 10% to 15%, 12.5% to 15%,10% to 30%, 10% to 40% or 10% to 50% increased risk for coronary heartdisease. In some embodiments, the human subject is homozygous for aSNP(s). In certain embodiments, the human subject that is homozygous forthe SNP(s) has a 10% to 30%, 10% to 25% or 10% to 20% greater likelihoodof developing coronary heart disease than a human subject that isheterozygous for the SNP(s). In other embodiments, the human subject isheterozygous for a SNP(s). The presence of the allele in a human subjectcan be detected using an in vitro assay, e.g., a nucleic acid sample(see Section 5.3 below) and techniques known to one of skill in the artor described in Section 5.4 below.

In another embodiment, the invention provides a method for determiningwhether a human subject carries a SNP, insertion and/or deletionassociated with an increased risk for having a myocardial infarction,comprising detecting the whether at least one, two, three, four or moreor all of the SNPs, insertions and/or deletions listed in Table 7, Table8, Table 10 or Table 11 is present in the human subject. In someembodiments, the human subject is identified as having or determined tohave an increased risk for having a myocardial infarction if one, two,three four or more, all or any combination the SNPs, insertions and/ordeletions is/are detected. In a specific embodiment, the presence ofone, two, three four or more, all or any combination of the SNPs,insertions and/or deletions listed in Table 7, Table 8, Table 10 orTable 11 indicates that the human subject has a 10% to 25%, 10% to 20%,10% to 15%, 12.5% to 15%, 10% to 30%, 10% to 40% or 10% to 50% increasedrisk for having a myocardial infarction. In some embodiments, the humansubject is homozygous for a SNP(s). In certain embodiments, the humansubject that is homozygous for the SNP(s) has a 10% to 30%, 10% to 25%or 10% to 20% greater likelihood of having a myocardial infarction thana human subject that is heterozygous for the SNP(s). In otherembodiments, the human subject is heterozygous for a SNP(s).

In certain embodiments, in addition to detecting one or more SNPs,insertions and/or deletions listed in Table 7, Table 8, Table 10 orTable 11, one or more polymorphisms identified as being associated withan increased likelihood of coronary heart disease (e.g., coronaryatherosclerosis or myocardial infarction), such as those described inInternational Publication No. WO 2007/006862, WO 2004/03576, WO2004/035741, and WO 2006/105439; Shiffman et al (2005) Am J Hum Genet77: 596-605; Shiffman et at (2006) Arteriosclerosis, Thrombosis, andVascular Biology 26: 1613-1618; Iakoubova et al. (2006)Arteriosclerosis, Thrombosis, and Vascular Biology 26: 2763-2768;Morrison et al. (2007) American Journal of Epidemiology 166: 28-35; Lukeet al. (2007) Arteriosclerosis, Thrombosis, and Vascular Biology 27:2030-2036; Shiffman et al. (2008) Arteriosclerosis, Thrombosis, andVascular Biology 28: 173-179; Iakoubova ea al. (2008) J Am Coll Cardiol51: 449-455; Iakoubova et al. (2008) J Am Coll Cardiol 51: 435-443;Shiffman et al. (2008) J Am Coll Cardiol 51:444-448; Bare et al. (2007)Genetics in Medicine 9: 682-689; Helgadottir et alt (2004) Nat. Genet.36: 233-239; and U.S. Patent Application Publication Nos. 2007/0280917,2006/0019269, 2005/0282855, 2005/0164220, 2005/0113408, 2005/0112611,2007/0031147, 2006/0228715, 2006/0223093, 2007/0072821 and 2005/0272054(each of which are incorporated by herein by reference) is alsodetected.

In one embodiment, the invention provides a method for determiningwhether a human subject carries a SNP, insertion and/or deletionassociated with increased risk for coronary heart disease (e.g.,coronary atherosclerosis or myocardial infarction), comprising detectingwhether at least one, two, three, four or more, all or any combinationof the following SNPs, insertions and/or deletions on human chromosome 9are present in the human subject: a guanine nucleotide at position22062264; a cytosine nucleotide at position 22062301; an adeninenucleotide at position 22062638; a guanine nucleotide at position22062719; a thymine nucleotide at position 22071397; a thyminenucleotide position 22071850; a thymine nucleotide position 22078090; aguanine nucleotide at position 22078094; a thymine nucleotide position22078260; a deletion at position 22078465; a guanine nucleotide atposition 22086055; a guanine nucleotide at position 22088574; a guaninenucleotide at position 22088619; an insertion at position 22089755; acytosine nucleotide position 22090176; a cytosine nucleotide at position22091702; a thymine nucleotide at position 22092165; a thyminenucleotide at position 22093183; a guanine nucleotide at position22093341; a guanine nucleotide at position 22093813; a cytosinenucleotide at position 22095927; an adenine nucleotide at position22096731; a cytosine nucleotide at position 22100131; an insertion atposition 22101587; a cytosine nucleotide at position 22102241; a guaninenucleotide at position 22102427; a cytosine nucleotide at position22104469; a guanine nucleotide at position 22104495; a guaninenucleotide at 22105026; a cytosine nucleotide at position 22105286; aguanine nucleotide at position 22106046; a cytosine nucleotide atposition 22106220; an insertion at position 22110491; a cytosinenucleotide at position 22113766; an adenine nucleotide at position22114123; a thymine nucleotide at position 22114140; a cytosinenucleotide at position 22115347; and/or a cytosine at position 22115503.In some embodiments, the human subject is identified as having ordetermined to have an increased risk for coronary heart disease if one,two, three four or more, all or any combination the SNPs, insertionsand/or deletions is/are detected. In a specific embodiment, the humansubject is has an increased risk for coronary heart disease. In aspecific embodiment, the presence of one, two, three four or more, allor any combination of the SNPs, insertions and/or deletions listed inTable 7, Table 8, Table 10 or Table 11 indicates that the human subjecthas a 10% to 25%, 10% to 20%, 10% to 15%, 12.5% to 15%, 10% to 30%, 10%to 40% or 10% to 50% increased risk for having a myocardial infarction.In some embodiments, the human subject is homozygous for a SNP(s). Incertain embodiments, the human subject that is homozygous for the SNP(s)has a 10% to 30%, 10% to 25% or 10% to 20% greater likelihood fordeveloping coronary heart disease than a human subject that isheterozygous for the SNP(s). In other embodiments, the human subject isheterozygous for a SNP(s). The presence of SNPs in a human subject canbe detected using an in vitro assay, e.g., a nucleic acid sample (seeSection 5.3 below) and techniques known to one of skill in the art ordescribed in Section 5.4 below.

In another embodiment, the invention provides a method for determiningwhether a human subject has a SNP associated with an increased risk forcoronary heart disease (e.g., coronary atherosclerosis or myocardialinfarction), comprising detecting whether a guanine nucleotide atposition 22086055 (rs10757274) and/or a guanine nucleotide at position22105026 (rs2383206) of human chromosome 9 is/are present in the human,and at least one, two, three, or more, all or any combination of thefollowing SNPs on human chromosome 9: a cytosine nucleotide at position22062301 (rs9632884), a thymine nucleotide at position 22071850(rs6475606), a thymine nucleotide at position 22078260 (rs10757272), aguanine at position 22088574 (rs4977574), a guanine nucleotide atposition 22088619 (rs2891168), a guanine nucleotide at position 22093813(rs1333042), a cytosine nucleotide at position 22115347 (rs1333048),and/or a cytosine nucleotide at position 22115503 (rs1333049). In aspecific embodiment, the human subject is has an increased risk ofmyocardial infarction. In some embodiments, the human subject isidentified as having or determined to have an increased risk for havinga coronary heart disease if two, three four or more, all or anycombination the SNPs are detected. In a specific embodiment, thepresence of two, three four or more, all or any combination of the SNPslisted in Table 7, Table 8, Table 10 or Table 11 indicates that thehuman subject has a 10% to 25%, 10% to 20%, 10% to 15%, 12.5% to 15%,10% to 30%, 10% to 40% or 10% to 50% increased risk for having amyocardial infarction. In some embodiments, the human subject ishomozygous for a SNP(s). In certain embodiments, the human subject thatis homozygous for the SNP(s) has a 10% to 30%, 10% to 25% or 10% to 20%greater likelihood of having a myocardial infarction than a humansubject that is heterozygous for the SNP(s). In other embodiments, thehuman subject is heterozygous for a SNP(s). The presence of SNPs in ahuman subject can be detected using an in vitro assay, e.g., a nucleicacid sample (see Section 5.3 below) and techniques known to one of skillin the art or described in Section 5.4 below.

In another embodiment, the invention provides a method for determiningwhether a human subject has a SNP associated with increased risk forcoronary heart disease (e.g., coronary atherosclerosis or myocardialinfarction), comprising detecting whether a guanine at position 22086055(rs10757274) and a guanine at position 22105026 (rs2383206) of humanchromosome 9 is present in the human subject. In some embodiments, thehuman subject is identified as having or determined to have an increasedrisk for developing coronary heart disease if the SNPs are detected. Ina specific embodiment, the human subject is has an increased risk ofmyocardial infarction. In some embodiments, the human subject that ishomozygous at positions 22086055 and 22105026 (i.e., has two guaninealleles at those positions) has a greater likelihood for developingcoronary heart disease than a human subject heterozygous at thosepositions. In other embodiments, the human subject is heterozygous atposition 22086055 and/or at position 22105026 of chromosome 9.

In another embodiment, the invention provides a method for determiningwhether a human subject has a SNP associated with increased risk forcoronary heart disease (e.g., coronary atherosclerosis or myocardialinfarction), comprising detecting whether a cytosine at position22115503 (rs1333049) of human chromosome 9 is present in the humansubject. In some embodiments, the human subject is identified as havingor determined to have an increased risk for developing coronary heartdisease if the SNP is detected. In a specific embodiment, the humansubject is has an increased risk of myocardial infarction. In someembodiments, the human subject that is homozygous at positions 22115503(rs1333049) (i.e., has two cytosine nucleotides at position 22115503(rs1333049)) has a greater likelihood for developing coronary heartdisease than a human subject heterozygous at that position 22115503(rs1333049) of human chromosome 9. In other embodiments, the humansubject is heterozygous at position 22115503 (rs1333049) of humanchromosome 9.

In another embodiment, the invention provides a method for determiningwhether a human subject has a SNP associated with increased risk forcoronary heart disease (e.g., coronary atherosclerosis or myocardialinfarction), comprising detecting whether a guanine at position 22114477(rs10757278) of human chromosome 9 is present in the human subject. Insome embodiments, the human subject is identified as having ordetermined to have an increased risk for developing coronary heartdisease if the SNP is detected. In a specific embodiment, the humansubject is has an increased risk of myocardial infarction. In someembodiments, the human subject that is homozygous at positions 22114477(rs10757278) (i.e., has two guanine nucleotides at position 22114477(rs1075728) has a greater likelihood for developing coronary heartdisease than a human subject heterozygous at position 22114477(rs10757278) of human chromosome 9. In other embodiments, the humansubject is heterozygous at position 22114477 (rs10757278) of humanchromosome 9.

In another embodiment, the invention provides a method for determiningwhether a human subject has a SNP associated with increased risk forcoronary heart disease (e.g., coronary atherosclerosis or myocardialinfarction), comprising detecting whether a cytosine at position22114477 (rs1333049) and a guanine at position 22114477 (rs10757278) ofhuman chromosome 9 are present in the human subject. In someembodiments, the human subject is identified as having or determined tohave an increased risk for developing coronary heart disease if the SNPsare detected. In a specific embodiment, the human subject is has anincreased risk of myocardial infarction, in some embodiments, the humansubject that is homozygous at position 22114477 (rs1333049) (i.e., twocytosine nucleotides at rs1333049) and homozygous at position 22114477(rs10757278) (i.e., two guanine nucleotides at rs10757278) has a greaterlikelihood for developing coronary heart disease than a human subjectheterozygous at position 22114477 (rs1333049) and at position 22114477(rs10757278) of human chromosome 9. In other embodiments, the humansubject is heterozygous at position 22114477 (rs1333049) and/or atposition 22114477 (rs10757278) of human chromosome 9.

In one embodiment, the invention provides a method for determiningwhether a human subject has an increased risk for coronary heart disease(e.g., coronary atherosclerosis or myocardial infarction), comprisingdetecting whether at least one, two, three, four or more or all of theSNPs, insertions and/or deletions listed in Table 7, Table 8, Table 10or Table 11 is present in a human subject, wherein the presence of one,two, three, four or more or all of the SNPs, insertions and/or deletionsindicates that the human subject has an increased risk for coronaryheart disease. In a specific embodiment, the human subject is has anincreased risk of myocardial infarction. The presence of SNPs,insertions and/or deletions in a human subject can be detected using anin vitro assay, e.g., a nucleic acid sample (see Section 5.3 below) andtechniques known to one of skill in the art or described in Section 5.4below. In a specific embodiment, the presence of one, two, three four ormore or all SNPs, insertions and/or deletions listed in Table 7, Table8, Table 10 or Table 11 indicates that the human subject has a 10% to25%, 10% to 20%, 10% to 15%, 12.5% to 15%, 10% to 30%, 10% to 40% or 10%to 50% increased risk for coronary heart disease.

In certain embodiments, in addition to detecting one or more SNPs,insertions and/or deletions listed in Table 7, Table 8, Table 10 orTable 11, one or more polymorphisms identified as being associated withan increased likelihood of coronary heart disease, such as thosedescribed in International Publication No. WO 2007/006862, WO2004/03576, WO 2004/035741, and WO 2006/105439; Shiffman et al (2005) AmJ Hum Genet 77: 596-605; Shiffman et al (2006) Arteriosclerosis,Thrombosis, and Vascular Biology 26: 1613-1618; Iakoubova et al. (2006)Arteriosclerosis, Thrombosis, and Vascular Biology 26: 2763-2768;Morrison et al. (2007) American Journal of Epidemiology 166: 28-35; Lukeet al. (2007) Arteriosclerosis, Thrombosis, and Vascular Biology 27:2030-2036; Shiffman et al. (2008) Arteriosclerosis, Thrombosis, andVascular Biology 28: 173-179; Iakoubova et al. (2008) J Am Coll Cardiol51: 449-455; Iakoubova et al. (2008) J Am Coll Cardiol 51: 435-443;Shiffman et al. (2008) J Am Coll Cardiol 51:444-448; Bare et al. (2007)Genetics in Medicine 9: 682-689; Helgadottir et al. (2004) Nat. Genet.36: 233-239; and U.S. Patent Application Publication Nos. 2007/0280917,2006/0019269, 2005/0282855, 2005/0164220, 2005/0113408, 2005/0112611,2007/0031847, 2006/0228715, 2006/0223093, 2007/0072821 and 2005/0272054(each of which are incorporated by herein by reference) is alsodetected.

In one embodiment, the invention provides a method for determiningwhether a human subject has an increased risk for coronary heart disease(e.g., coronary atherosclerosis or myocardial infarction), comprisingdetecting whether at least one, two, three, four or more, all or anycombination of the following SNPs, insertions and/or deletions on humanchromosome 9 is/are present in a human subject: a guanine nucleotide atposition 22062264; a cytosine nucleotide at position 22062301; anadenine nucleotide at position 22062638; a guanine nucleotide atposition 22062719; a thymine nucleotide at position 22071397; a thyminenucleotide position 22071850; a thymine nucleotide position 22078090; aguanine nucleotide at position 22078094; a thymine nucleotide position22078260; a deletion at position 22078465; a guanine nucleotide atposition 22086055; a guanine nucleotide at position 22088574; a guaninenucleotide at position 22088619; an insertion at position 22089755; acytosine nucleotide position 22090176; a cytosine nucleotide at position22091702; a thymine nucleotide at position 22092165; a thyminenucleotide at position 22093183; a guanine nucleotide at position22093341; a guanine nucleotide at position 22093813; a cytosinenucleotide at position 22095927; an adenine nucleotide at position22096731; a cytosine nucleotide at position 22100131; an insertion atposition 22101587; a cytosine nucleotide at position 22102241; a guaninenucleotide at position 22102427; a cytosine nucleotide at position22104469; a guanine nucleotide at position 22104495; a guaninenucleotide at 22105026; a cytosine nucleotide at position 22105286; aguanine nucleotide at position 22106046; a cytosine nucleotide atposition 22106220; an insertion at position 22110491; a cytosinenucleotide at position 22113766; an adenine nucleotide at position22114123; a thymine nucleotide at position 22114140; a cytosinenucleotide at position 22115347; and/or a cytosine at position 22115503,wherein the presence of one, two, three, four or more, all or anycombination of the SNPs, insertions and/or deletions indicates that thehuman subject has an increased risk for coronary heart disease. In oneembodiment, the SNP is a cytosine at position 22115503 (rs1333049). In aspecific embodiment, the human subject is has an increased risk ofmyocardial infarction. In a specific embodiment, the human subject has a10% to 30%, 10% to 25% or 10% to 20% or a 10%, 15%, 25% or 30% greaterlikelihood of developing coronary heart disease if one, two, three, fouror more, all or any combination of the SNPs, insertions and/or deletionsare detected than a human subject in which such SNPs, insertions and/ordeletions are not detected. The presence of SNPs in a human subject canbe detected using an in vitro assay, e.g., a nucleic acid sample (seeSection 5.3 below) and techniques known to one of skill in the art ordescribed in Section 5.4 below.

In another embodiment, the invention provides a method for determiningwhether a human subject has an increased risk for coronary heart disease(e.g., coronary atherosclerosis or myocardial infarction), comprisingdetecting whether a guanine nucleotide at position 22086055 (rs10757274)and/or a guanine nucleotide at position 22105026 (rs2383206) of humanchromosome 9 is/are present in a human subject, and at least one, two,three, or more, all or any combination of the following SNPs on humanchromosome. 9: a cytosine nucleotide at position 22062301 (rs9632884), athymine nucleotide at position 22071850 (rs6475606), a thyminenucleotide at position 22078260 (rs10757272), a guanine at position22088574 (rs4977574), a guanine nucleotide at position 22088619(rs2891168), a guanine nucleotide at position 22093813 (rs1333042), acytosine nucleotide at position 22115347 (rs1333048), and/or a cytosinenucleotide at position 22115503 (rs333049), wherein the presence of one,two, three, four or more or all of the SNPs indicates that the humansubject has an increased risk for coronary heart disease. In a specificembodiment, the human subject is has an increased risk of myocardialinfarction. The presence of SNPs in a human subject can be detectedusing an in vitro assay, e.g., a nucleic acid sample (see Section 5.3below) and techniques known to one of skill in the art or described inSection 5.4 below.

In another embodiment, the invention provides a method for determiningwhether a human subject has an increased risk for coronary heart disease(e.g., coronary atherosclerosis or myocardial infarction), comprisingdetecting whether a guanine at position 22086055 (rs10757274) and aguanine at position 22105026 (rs2383206) of human chromosome 9 arepresent in human subject, wherein the presence of a guanine at thosepositions indicates that the human subject has an increased risk forcoronary heart disease. In a specific embodiment, the human subject ishas an increased risk of myocardial infarction. In some embodiments, thehuman subject is homozygous at positions 22086055 and 22105026. In aspecific embodiment, a human subject that is homozygous at positions22086055 and 22105026 (i.e., has two guanine nucleotides at thosepositions) has a greater likelihood for developing coronary heartdisease than a human subject heterozygous at those positions. In otherembodiments, the human subject is heterozygous at positions 22086055 and22105026.

In another aspect, the invention provides a method for determiningwhether a human subject has a polymorphism associated with an increasedrisk for coronary heart disease (e.g., coronary atherosclerosis ormyocardial infarction), comprising detecting whether one or more SNPs inan approximately 58 kb region extending from about 22,062,301 to about22,120,389 of human chromosome 9 with a p value of less than 0.025 orless than 0.050 (e.g., 0.025, 0.050 or 0.075) is/are present, whereinthe presence of the one or more SNPs indicates that the human subjecthas a polymorphism associated with an increased risk for coronary heartdisease. In some embodiments, the human subject is identified as havingor determined to have an increased risk for coronary heart disease ifthe one or more SNPs are detected. In some embodiments, the SNP is in aregion extending from position 22,062,301+/−5808, 5000, 4407, 4000,3000, 2500, 2000, 1500, 1101, 1000, 550, 500, 250, 200, 150, 100, or 50nucleotides to position 22,120,389+/−5808, 5000, 4407, 4000, 3000, 2500,2000, 1500, 1101, 1000, 550, 500, 250, 200, 150, 100 or 50 nucleotidesof human chromosome 9. In a specific embodiment, the SNP is in a regionextending from position 22,062,301 to position 22,120,389 of humanchromosome 9. In certain embodiments, in addition to detecting one ormore SNPs in a region extending from 22,062,301 to 22,120,389 of humanchromosome 9 with a p value of less than 0.025 or less than 0.050, oneor more polymorphisms identified as being associated with an increasedlikelihood of coronary heart disease, such as those described inInternational Publication No. WO 2007/006862, WO 2004/03576, WO2004/035741, and WO 2006/105439; Shiffman et al (2005) Am J Hum Genet77: 596-605; Shiffman et al (2006) Arteriosclerosis, Thrombosis, andVascular Biology 26: 1613-1618; Iakoubova at al. (2006)Arteriosclerosis, Thrombosis, and Vascular Biology 26: 2763-2768;Morrison t al. (2007) American Journal of Epidemiology 166: 28-35; Lukeat al. (2007) Arteriosclerosis, Thrombosis, and Vascular Biology 27:2030-2036; Shiffman et al. (2008) Arteriosclerosis, Thrombosis, andVascular Biology 28: 173-179; Iakoubova et al. (2008) J Am Coll Cardiol51: 449-455; Iakoubova et al. (2008) J Am Coll Cardiol 51: 435-443;Shiffman et al. (2008) J Am Coll Cardiol 51:444-448; Bare et al. (2007)Genetics in Medicine 9: 682-689; Helgadottir at al. (2004) Nat. Genet.36: 233-239; and U.S. Patent Application Publication Nos. 2007/0280917,2006/0019269, 2005/0282855, 2005/0164220, 2005/0113408, 2005/0112611,2007/0031847, 2007/0072821, 2006/0228715, 2006/0223093, and2005/0272054, each of which are incorporated herein by reference, isalso detected. In a specific embodiment, the human subject has anincreased risk of myocardial infarction. The presence of the SNPs can bedetected using an in vitro assay, e.g., a nucleic acid sample (Section5.3 below) and techniques known to one of skill in the art or describedin Section 5.4 below. In certain embodiments, the presence of one ormore or any combination of the SNPs listed in Table 7, Table 8, Table 10and/or Table 11 are detected. In certain embodiments, the term“significantly associated” refers to a statistically significantassociation between coronary heart disease and an allele, a haplotype ora SNP.

In certain embodiments, a person identified as having an increased riskfor coronary heart disease is administered a therapy to delay the onset,prevent the onset or slow the progression of coronary heart disease. Insome embodiments, a person identified as having an increased risk for amyocardial infarction is administered a therapy to delay or prevent theonset of a myocardial infarction. In particular embodiments, a personcharacterized for increased risk for coronary atherosclerosis is treatedwith a therapy to delay the onset, prevent the onset or slow progressionof the coronary atherosclerosis, particularly wherein the therapycomprises a lipid-lowering medication. Non-limiting examples oftherapies that may be administered to delay the onset, prevent the onsetor slow the progression of coronary heart disease (including myocardialinfarction and coronary atherosclerosis), include: aspirin, digitalis,ACE (angiotensin converting enzyme) inhibitors, beta blockers, nitrates(such as nitroglycerine), calcium-channel blockers, diuretics, bloodcholesterol-lowering agents, and thrombolytic agents. In certainembodiments, a person identified as having an increased risk forcoronary heart disease is administered a currently available therapy toprevent or treat heart disease or a therapy that will be developed oridentified as having utility to prevent or treat coronary heart disease.In some embodiments, the SNPs, alleles, haplotypes, and otherpolymorphisms described herein are used to assess a person'sresponsiveness to a therapy for coronary heart disease.

In some embodiments, a person identified as having an increased risk forcoronary heart disease adopts one or more lifestyle changes alone or incombination with receiving one or more therapies to delay the onset,prevent the onset, or slow the progression of coronary heart disease(including myocardial infarction and coronary atherosclerosis).Non-limiting examples of lifestyle changes include: stopping smokingcigarettes, towering high blood pressure, reducing high bloodcholesterol, losing extra weight, becoming physically active, andmanaging diabetes.

5.2. Characteristics of Human Subjects

In certain embodiments, the human subject evaluated for an increasedrisk for coronary heart disease in accordance with the methods of theinvention is a human adult. In some embodiments, the human subjectevaluated for an increased risk for coronary heart disease in accordancewith the methods of the invention is an elderly human. In someembodiments, the human subject evaluated for an increased risk forcoronary heart disease in accordance with the methods of the inventionis a human infant. In some embodiments, the human subject evaluated foran increased risk for coronary heart disease in accordance with themethods of the invention is a human child or human toddler. Inparticular embodiments, the human subject evaluated for an increasedrisk for coronary heart disease in accordance with the methods of theinvention is a 40 to 85 year old human, a 50 to 85 year old human, a 50to 75 year old human, a 50 to 70 year old human, a 50 to 65 year oldhuman, a 55 to 85 year old human, a 55 to 80 year old human, a 55 to 75year old human, a 55 to 65 year old human, a 71.1 to 79.7 year oldhuman, a 45 to 64 year old human, a 48 to 60 year old human or a 42 to72 year old human. In specific embodiments, the human subject evaluatedfor an increased risk for coronary heart disease in accordance with themethods of the invention is a 45 year old human, a 50 year old human, a53 year old, a 54 year old human, a 55 year old human, a 56 year oldhuman, a 57 year old human, a 58 year old human, a 59 year old human, a60 year old human, a 65 year old human, a 70 year old human, a 72 yearold human, a 75 year old human, an 80 year old human or an 85 year oldhuman.

In certain embodiments, the human subject evaluated for an increasedrisk for coronary heart disease in accordance with the methods of theinvention is a female. In other embodiments, the human subject evaluatedfor an increased risk for coronary heart disease in accordance with themethods of the invention is a male.

In certain embodiments, the human subject evaluated for an increasedrisk for coronary heart disease in accordance with the methods of theinvention is a black, a white, an Indian or an Asian. In someembodiments, the human subject evaluated for an increased risk forcoronary heart disease in accordance with the methods of the inventionis Caucasian. In certain embodiments, the human subject evaluated for anincreased risk for coronary heart disease in accordance with the methodsof the invention is an African American. In other embodiments, the humansubject evaluated for an increased risk for coronary heart disease inaccordance with the methods of the invention is not African American. Insome embodiments, the human subject evaluated for an increased risk forcoronary heart disease in accordance with the methods of the inventionis Asian.

In certain embodiments, the human subject evaluated for an increasedrisk for coronary heart disease in accordance with the methods of theinvention smokes cigarettes. In some embodiments, the human subjectevaluated for an increased risk for coronary heart disease in accordancewith the methods of the invention has a triglyceride level of between150 to 199 mg/dL, 200 to 499 mg/dL, or 500 mg/dL or more. In particularembodiments, the human subject evaluated for an increased risk forcoronary heart disease in accordance with the methods of the inventionhas a triglyceride level of at least 175 mg/dL, at least 180 mg/dL, atleast 185 mg/dL, at least 190 mg/dL, at least 195 mg/dL, at least 200mg/dL, at least 225 mg/dL, or at least 250 md/dL. In certainembodiments, the human subject evaluated for an increased risk forcoronary heart disease in accordance with the methods of the inventionhas a triglyceride level of less than 150 mg/dL.

In certain embodiments, the human subject evaluated for an increasedrisk for coronary heart disease in accordance with the methods of theinvention has a total cholesterol level of between 200 to 239 mg/dL. Inother embodiments, the human subject evaluated for an increased risk forcoronary heart disease in accordance with the methods of the inventionhas a total cholesterol level of 240 mg/dL or more. In yet otherembodiments, the human subject evaluated for an increased risk forcoronary heart disease in accordance with the methods of the inventionhas a total cholesterol level of less than 200 mg/dL or less than 280mg/dL.

In some embodiments, the human subject evaluated for an increased riskfor coronary heart disease in accordance with the methods of theinvention has been diagnosed as having diabetes. In other embodiments,the human subject evaluated for an increased risk for coronary heartdisease in accordance with the methods of the invention does not have orhas yet to be diagnosed as having diabetes. In specific embodiments, thehuman subject evaluated for an increased risk for coronary heart diseasein accordance with the methods of the invention has a total cholesterollevel of less than 280 mg/dL and does not have or has not been diagnosedwith diabetes.

In certain embodiments, the human subject evaluated for an increasedrisk for coronary heart disease in accordance with the methods of theinvention has hypertension. In certain embodiments, the human subjectevaluated for an increased risk for coronary heart disease in accordancewith the methods of the invention has a normal body mass index (BMI) of18.5-24.9. In other embodiments, the human subject evaluated for anincreased risk for coronary heart disease in accordance with the methodsof the invention has an overweight BMI of 25-29.9. In yet otherembodiments, the human subject evaluated for an increased risk forcoronary heart disease in accordance with the methods of the inventionhas an obese BMI of 30 or more.

In some embodiments, the human subject evaluated for an increased riskfor coronary heart disease in accordance with the methods of theinvention has a family history of coronary heart disease. In oneembodiment, the human subject evaluated for an increased risk forcoronary heart disease in accordance with the methods of the inventionhas a family history of coronary atherosclerosis. In another embodiment,the human subject evaluated for an increased risk for coronary heartdisease in accordance with the methods of the invention has a familyhistory of myocardial infarction. In another embodiment, the humansubject evaluated for an increased risk for coronary heart disease inaccordance with the methods of the invention has a family history ofangina.

In certain embodiments, the human subject evaluated for an increasedrisk for coronary heart disease in accordance with the methods of theinvention do not have a history of cardiovascular symptoms. Non-limitingexamples of cardiovascular symptoms include a positive stress test,coronary angiography demonstrating stenosis (>50%) in any artery, andclinical cardiovascular events.

In certain embodiments, the human subject evaluated for an increasedrisk for coronary heart disease in accordance with the methods of theinvention has one, two, three or more characteristics of the populationof human subjects used in the studies discussed in the working example.In a specific embodiment, the human subject evaluated for an increasedrisk for coronary heart disease in accordance with the methods of theinvention has one, two, three or more characteristics of the populationof human subjects used in the Ottawa Heart Study discussed in theworking example. In another embodiment, the human subject evaluated foran increased risk for coronary heart disease in accordance with themethods of the invention has one, two, three or more characteristics ofthe population of human subjects used in the Atherosclerosis Risk inCommunities Study discussed in the working example. In anotherembodiment, the human subject evaluated for an increased risk forcoronary heart disease in accordance with the methods of the inventionhas one, two, three or more characteristics of the population of humansubjects used in the Copenhagen City Heart Study discussed in theworking example. In another embodiment, the human subject evaluated foran increased risk for coronary heart disease in accordance with themethods of the invention has one, two, three or more characteristics ofthe population of human subjects used in the Dallas Heart Study.

5.3. Biological Sample from Human Subjects for Screening

Samples for use in accordance with the methods of the invention includehuman biological samples comprising genomic DNA and/or RNA, and samplesisolated, obtained and/or derived from a human biological sample whichsamples comprise human genomic DNA, amplified genomic DNA, RNA,amplified RNA and/or cDNA (i.e., a nucleic acid sample). In certainembodiments, in addition to the biological sample itself or in additionto material derived from the biological sample, such as cells andgenomic DNA, the sample used in the methods of this invention comprises,for example, added water, salts, glycerin, glucose, an antimicrobialagent, paraffin, a chemical stabilizing agent, heparin, ananticoagulant, and/or a buffering agent.

A sample derived from a biological sample is one in which the biologicalsample has been subjected to one or more pretreatment steps prior to usein accordance with the methods of the invention. In certain embodiments,a biological fluid is pretreated by centrifugation, filtration,precipitation, dialysis, or chromatography, or by a combination of suchpretreatment steps. In other embodiments, a tissue sample is pretreatedby freezing, chemical fixation, paraffin embedding, dehydration,permeabilization, or homogenization followed by centrifugation,filtration, precipitation, dialysis, or chromatography, or by acombination of such pretreatment steps. In certain embodiments, thesample is pretreated by adjusting the concentration of nucleic acid inthe sample, by adjusting the pH or ionic strength of the sample, or byremoving contaminating proteins, nucleic acids, lipids, or debris fromthe sample.

A biological sample containing genomic DNA and/or RNA can be obtainedfrom any tissue or organ in a human subject. Representative biologicalsamples from a human subject include, without limitation, nasal swabs,throat swabs, dermal swabs, blood (including blood culture), buccalswabs, lymph, lung tissue, ejaculatory fluid, saliva, sputum, vaginalsecretions, stool, tears, spinal fluid, and synovial fluid. A biologicalsample can be stored (e.g., at approximately 22° C., 4° C., −20° C. orbelow −60° C.) before use.

In a specific embodiment, the biological sample is a blood sample. Ablood sample may be obtained from a subject according to methods wellknown in the art. Blood may be drawn from a subject from any part of thebody (e.g., a finger, a hand, a wrist, an arm, a leg, a foot, an ankle,a stomach, or a neck) using techniques known to one of skill in the art,in particular methods of phlebotomy known in the art. In a specificembodiment, venous blood is obtained from a subject and utilized inaccordance with the methods of the invention. In another embodiment,arterial blood is obtained and utilized in accordance with the methodsof the invention. For routine blood tests, venous blood is generallyused.

In some embodiments of the present invention, blood is collected and/orstored in a K₃/EDTA tube. In a specific embodiment, blood is collectedand/or stored in ACD-A tubes (Becton Dickinson Catalog No. 364606). Inanother embodiment, blood is collected and/or stored on one, two, three,four or more FAST TECHNOLOGY FOR ANALYSIS (FTA®) cards, such as FTA®Classic Cards, FTA® MINI CARDS, FTA® MICRO CARDS, and FTA® GENE CARDS(Whatman).

In some embodiments, the collected blood is stored prior to use. In oneembodiment, the collected blood is stored at room temperature (i.e.,approximately 22° C.). In another embodiment, the collected blood isstored at refrigerated temperatures, such as 4° C., prior to use. Insome embodiments, a portion of the blood sample is used in accordancewith the invention at a first instance of time whereas one or moreremaining portions of the blood sample is stored for a period of timefor later use. This period of time can be an hour or more, a day ormore, a week or more, a month or more, a year or more, or indefinitely.For long term storage, storage methods well known in the art, such asstorage at cryo temperatures (e.g. below −60° C.) can be used. In someembodiments, in addition to storage of the blood or instead of storageof the blood, isolated nucleic acids (e.g., isolated genomic DNA) isstored for a period of time for later use. Storage of such nucleic acidscan be for an hour or more, a day or more, a week or more, a month ormore, a year or more, or indefinitely.

In some embodiments of the present invention, blood cells are separatedfrom whole blood collected from a subject using techniques known in theart. For example, blood collected from a subject can be subjected toFicoll-Hypaque (Pharmacia) gradient centrifugation. Such centrifugationseparates erythrocytes (red blood cells) from various types of nucleatedcells and from plasma.

Blood cells can be sorted using a fluorescence activated cell sorter(FACS). Fluorescence activated cell sorting (FACS) is a known method forseparating particles, including cells, based on the fluorescentproperties of the particles. See, for example, Kamarch, 1987, MethodsEnzymol 151:150-165. Magnetic beads can also be used to separate bloodcells in some embodiments of the present invention. Separated bloodcells (e.g., leukocytes) can be frozen by standard techniques prior touse in the present methods.

In some embodiments, blood cells are immortalized and/or proliferated incell culture prior to use or storage. Any technique known in the art forimmortalizing and/or proliferating blood cells can be used in accordancewith the invention. In certain embodiments, the blood cells (e.g.,lymphocytes) are infected with a virus, such as HTLV-I or HTLV-II, thatimmortalizes the cells. In other embodiments, the blood cells aretransformed with an oncogene, such as bcl-2, that immortalizes thecells. In some embodiments, the blood cells are stored prior to or afterproliferation and/or immortalization. In one embodiment, the blood cellsare stored at cryo temperatures (e.g. below −60° C.).

In another embodiment, the biological sample collected from a humansubject is a swab of buccal cells from a subject's inner cheek (i.e., acheek or buccal swab). In another embodiment, the biological sample is atissue sample that comprises nucleated cells. In a particularembodiment, the tissue sample is breast, colon, lung, liver, ovarian,pancreatic, heart, prostate, renal, bone or skin tissue. In a specificembodiment, the tissue sample is a biopsy. Techniques for collectingbiological samples are known to those of skill in the art.

In some embodiments, the collected cheek swab or tissue sample is storedprior to use. In one embodiment, the collected cheek swab or tissuesample is stored at room temperature (e.g., approximately 22° C.). Inanother embodiment, the collected cheek swab or tissue sample is storedat refrigerated temperatures, such as 4° C., prior to use. In someembodiments, a portion of the tissue sample is used in accordance withthe invention at a first instance of time whereas one or more remainingportions of the tissue sample is stored for a period of time for lateruse. This period of time can be an hour or more, a day or more, a weekor more, a month or more, a year or more, or indefinitely. For long termstorage, storage methods well known in the art, such as storage at cryotemperatures (e.g. below −60° C.) can be used. In some embodiments, inaddition to storage of the cheek swab or tissue sample, or instead ofstorage of the cheek swab or tissue sample, isolated nucleic acids(e.g., isolated genomic DNA) is stored for a period of time for lateruse. Storage of such nucleic acids can be for an hour or more, a day ormore, a week or more, a month or more, a year or more, or indefinitely.

A tissue sample can be separated into cell types such as epithelialcells, fibroblasts, etc. and such cell types can be used in accordancewith the invention. In some embodiments, cells are immortalized and/orproliferated in cell culture prior to use or storage. Any techniqueknown in the art for immortalizing and/or proliferating cells can beused in accordance with the invention. In certain embodiments, the cells(e.g., lymphocytes) are infected with a virus that immortalizes thecells. In other embodiments, the cells are transformed with an oncogene,such as bcl-2, that immortalizes the cells. In some embodiments, thecells isolated from a cheek swab or tissue sample are stored prior to orafter proliferation and/or immortalization. In one embodiment, the cellsare stored at cryo temperatures (e.g. below −60° C.).

In some embodiments, after a biological sample is obtained from a humansubject, the biological sample is subjected to one or pretreatmentsteps. In one embodiment, after a biological sample is obtained from ahuman subject, genomic DNA is extracted. In another embodiment, after abiological sample is obtained from a human subject, cells (e.g., cellsof a particular type, such as T-lymphocytes) are cultured and thegenomic DNA from the cultured cells is extracted. In some embodiments,extracted genomic DNA is stored (e.g., at approximately 4° C. or below−60° C.) before use. In certain embodiments, extracted genomic DNA isamplified before use in accordance with the methods of the invention.Any technique known to one of skill in the art may be used culturecells, to extract genomic DNA and to amplify genomic DNA.

There are several known methods for extracting genomic DNA frombiological samples, any of which can be used in the present invention.One nonlimiting example follows. Between 60-80 mg of tissue is placed ina petri dish with culture media and the tissue is divided into twopieces. The tissue is placed into two sterile 15 ml tubes andcentrifuged for two minutes at 4° C. at 1500 rpm. The supernatant isremoved and washed twice with 1 ml 1×PBS or DNA-buffer. The supernatantis removed the pellet resuspended in 2.06 ml DNA-buffer. About 100 μl ofproteinase K (10 mg/ml) and 240 μl 10% SDS is added, and the solution isshaken gently before incubation overnight at 45° C. in a waterbath. Ifthere are still some tissue pieces visible, proteinase K is added again,the solution shaken gently, and incubated for another 5 hr at 45° C.About 2.4 ml of phenol is then added and the solution is shaken by handfor 5-10 minutes before centrifugation at 3000 rpm for 3 minute at 10°C. The supernatant is pipetted into a new tube, 1.2 ml of phenol isadded, 1.2 ml of chloroform/isoamyl alcohol (24:1) is added and then thesolution is shaken by hand for 5-10 min before centrifugation at 3000rpm for 5 minute at 10° C. The supernatant is pipetted into a new rubeand 2.4 ml of chloroform/isoamyl alcohol (24:1) is added. The solutionis shaken by hand for 5-10 minutes, and centrifuged at 3000 rpm for 5minutes at 10° C. The supernatant is pipetted into a new tube, 25 μl of3 M sodium acetate (pH 5.2) is added, 5 ml ethanol is added, and thenthe solution shaken gently until the DNA precipitates. A glass pipetteis heated over a gas burner and the end bent to a hook. The DNA threadis fished out of the solution using the hook and transferred to a newtube. The DNA is washed in 70% ethanol and dried in a speed vacuum. TheDNA is dissolved in 0.5-1 ml sterile water overnight (or longer ifnecessary) at 4° C. on a rotating shaker.

In some embodiments, after a biological sample containing RNA isobtained from a human subject, the biological sample is subjected to oneor more pretreatment steps and the RNA is extracted. In certainembodiments, the RNA is amplified or used to produce cDNA. In certainembodiments, the RNA is stored (e.g., at approximately 4° C., 30° C. or−70° C.) before use. Any technique known to one of skill in the art maybe used to extract RNA, to amplify and to produce cDNA.

Techniques for isolating nucleic acids include, e.g., those taught inBerger and Kimmel, Guide to Molecular Cloning Techniques. Methods inEnzymology volume 152 Academic Press, Inc., San Diego, Calif. (Berger):Sambrook et al., Molecular Cloning—A Laboratory Manual (3rd Ed.), Vol.1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 2001(“Sambrook”); and/or Current Protocols in Molecular Biology, F. M.Ausubel et alt, eds., Current Protocols, a joint venture between GreenePublishing Associates, Inc. and John Wiley & Sons, Inc. (supplementedthrough 2002) (“Ausubel”)). A plethora of kits are also commerciallyavailable for the purification of nucleic acids from cells or othersamples (see, e.g., EasyPrep™, FlexiPrep™, both from Pharmacia Biotech;StrataClean™, from Stratagene; and, QIAprep™ from Qiagen).

In some embodiments, a genomic DNA sample, an amplified genomic DNAsample, a RNA sample, an amplified RNA and/or a cDNA is stored (e.g., atapproximately 4° C., −30° C. or −70° C.) before use. Techniques forstoring such samples are known to one of skill in the art.

In specific embodiments, a sample comprising genomic DNA, amplified DNA,RNA, amplified RNA and/or cDNA isolated, obtained and/or derived from abiological sample from a human subject is added to a buffer to make asuspension and an aliquot of the suspension is used in accordance withthe methods of the invention. In some embodiments, a sample ofcomprising genomic DNA, amplified DNA. RNA, amplified RNA and/or cDNAisolated, obtained and/or derived from a biological sample from a humansubject is added to sterile water to make a suspension and an aliquot ofthe suspension is used in accordance with the methods of the invention.

5.4. Techniques for Detecting Polymorphic Sites

Polymorphisms may be detected using any established method available inthe art, including, without limitation, Southern blotting, allelespecific hybridization (ASH), detection of single nucleotide extension,sequencing, array hybridization (optionally including ASH), amplifiedfragment length polymorphism (AFLP) detection, amplified variablesequence detection, randomly amplified polymorphic DNA (RAPD) detection,restriction fragment length polymorphism (RFLP) detection,self-sustained sequence replication detection, simple sequence repeat(SSR) detection, single-strand conformation polymorphisms (SSCP)detection, northern analysis, quantitative amplification of mRNA orcDNA, or the like. Any of these techniques are readily adapted to highthroughput analysis.

In one embodiment, the presence or absence of a polymorphism isdetermined by genotyping or nucleotide sequencing using techniques knownto one of skill in the art. Exemplary genotyping methods are describedin Chen et al., “Single nucleotide polymorphism genotyping:biochemistry, protocol, cost and throughput”, Pharmacogenomics J. 2003;3(2):77-96; Kwok et al., “Detection of single nucleotide polymorphisms”,Curr Issues Mol Biol. 2003 April; 5(2):43-60; Shi, “Technologies forindividual genotyping: detection of genetic polymorphisms in drugtargets and disease genes”, Am J Pharmacogenomics. 2002; 2(3):197-205;and Kwok, “Methods for genotyping single nucleotide polymorphisms”, AnnuRev Genomics Hum Genet 2001; 2:235-58. Exemplary techniques forhigh-throughput SNP genotyping are described in Marnellos,“High-throughput SNP analysis for genetic association studies”, CurrOpin Drug Discov Devel. 2003 May; 6(3):317-21. Common SNP genotypingmethods include, but are not limited to, TaqMan assays, molecular beaconassays, nucleic acid arrays, allele-specific primer extension,allele-specific PCR, arrayed primer extension, homogeneous primerextension assays, primer extension with detection by mass spectrometry,pyrosequencing, multiplex primer extension sorted on genetic arrays,ligation with rolling circle amplification, homogeneous ligation, OLA(U.S. Pat. No. 4,988,167), multiplex ligation reaction sorted on geneticarrays, restriction-fragment length polymorphism, single baseextension-tag assays, and the Invader assay. For example, massspectrometry may be used for genotyping. Mass spectrometry takesadvantage of the unique mass of each of the four nucleotides of DNA.Polymorphisms can be unambiguously genotyped by mass spectrometry bymeasuring the differences in the mass of nucleic acids havingalternative alleles. MALDI-TOF (Matrix Assisted Laser DesorptionIonization-Time of Flight) mass spectrometry technology is preferred forextremely precise determinations of molecular mass. The followingreferences provide information describing mass spectrometry-basedmethods for SNP genotyping: Bocker, “SNP and mutation discovery usingbase-specific cleavage and MALDI-TOF mass spectrometry”, Bioinformatics.2003 July; 9 Suppl 1:144-153; Storm t al., “MALDI-TOF massspectrometry-based SNP genotyping”, Methods Mol Biol 2003; 212241-62;Jurinke et al., “The use of MassARRAY technology for high throughputgenotyping”, Adv Biochem Eng Biotechnol. 2002; 77:57-74; and Jurinke etal., “Automated genotyping using the DNA MassArray technology”, MethodsMol Biol. 2002; 187:179-92.

Procedures for performing Southern blotting, standard amplification(PCR, LCR, or the like) and many other nucleic acid detection methodsare well established and are taught, e.g., in Sambrook et al., MolecularCloning—A Laboratory Manual (3rd Ed.), Vol. 1-3, Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y., 2000 (“Sambrook”); CurrentProtocols in Molecular Biology, F. M. Ausubel et al., eds., CurrentProtocols, a joint venture between Greene Publishing Associates, Inc.and John Wiley & Sons, Inc., (supplemented through 2002) (“Ausubel”))and PCR Protocols A Guide to Methods and Applications (Innis et al. eds)Academic Press Inc. San Diego, Calif. (1990) (Innis).

In some embodiments, a polymorphism is detected using unamplifiedgenomic DNA by, e.g., performing a Southern blot on a sample of genomicDNA. In other embodiments, a polymorphism is detected using amplifiedgenomic DNA or cDNA.

Some techniques for detecting polymorphisms utilize hybridization of anucleic acid probe to nucleic acids corresponding to the polymorphism(e.g., amplified nucleic acids produced using genomic DNA as atemplate). Hybridization formats, including, but not limited to:solution phase, solid phase, mixed phase, or in situ hybridizationassays are useful for allele detection. An extensive guide to thehybridization of nucleic acids is found in Tijssen (1993) LaboratoryTechniques in Biochemistry and Molecular Biology—Hybridization withNucleic Acid Probes Elsevier, New York, as well as in Sambrook, Bergerand Ausubel.

Briefly, restriction fragment length polymorphisms (RFLP) generallyinvolves hybridizing a probe which is typically a sub-fragment (or asynthetic oligonucleotide corresponding to a sub-fragment) of thenucleic acid to be detected to restriction digested genomic DNA. Therestriction enzyme is selected to provide restriction fragments of atleast two alternative (or polymorphic) lengths in different individualsor populations. Identifying one or more restriction enzymes thatproduces informative fragments for each allele of a marker is a simpleprocedure, well known in the art. After separation by length in anappropriate matrix (e.g., agarose or polyacrylamide) and transfer to amembrane (e.g., nitrocellulose, nylon, etc.), the labeled probe ishybridized under conditions which result in equilibrium binding of theprobe to the target followed by removal of excess probe by washing.

Some techniques for detecting polymorphisms comprise an amplificationstep. Examples of such techniques include, but are not limited to, thepolymerase chain reaction (PCR), reverse transcription PCR (RT-PCR), theligase chain reaction (LCR), Qβ-replicase amplification and other RNApolymerase mediated techniques (e.g., NASBA). For references describingsuch techniques see, e.g., Innis, Sambrook. Ausubel, and Berger.Additional details are found in Mullis et al. (1987) U.S. Pat. No.4,683,202; Arnheim & Levinson (Oct. 1, 1990) C&EN 36-47; The Journal OfNIH Research (1991) 3, 81-94; (Kwoh et al. (1989) Proc. Natl. Acad. Sci.USA 86, 1173; Guatelli et al. (1990) Proc. Natl. Acad. Sci USA 87, 1874;Lomell et al. (1989) J. Clin. Chem 35, 1826; Landegren et al., (1988)Science 241, 1077-1080; Van Brunt (1990) Biotechnology 8, 291-294; Wuand Wallace, (1989)Gene 4, 560; Barringer et al. (1990) Gene 89, 117,and Sooknanan and Malek (1995) Biotechnology 13: 563-564. Improvedmethods of amplifying large nucleic acids by PCR, which is useful in thecontext of positional cloning, are further summarized in Cheng et al.(1994) Nature 369: 684, and the references therein, in which PCRamplicons of up to 40 kb are generated. Methods for long-range PCR aredisclosed, for example, in U.S. Pat. No. 6,898,531, issued May 24, 2005,entitled “Algorithms for Selection of Primer Pairs”; U.S. patentapplication Ser. No. 10/236,480, filed Sep. 9, 2002, entitled “Methodsfor Amplification of Nucleic Acids”; and U.S. Pat. No. 6,740,510, issuedMay 25, 2004, entitled “Methods for Amplification of Nucleic Acids”.U.S. Ser. No. 10/341,832 (filed Jan. 14, 2003) also provides detailsregarding primer picking methods for performing short range PCR.

Real time PCR or LCR may be performed on amplification mixtures, e.g.,using molecular beacons or TaqMan™ probes. A molecular beacon (MB) is anoligonucleotide or PNA which, under appropriate hybridizationconditions, self-hybridizes to form a stem and loop structure. The MBhas a label and a quencher at the termini of the oligonucleotide or PNA;thus, under conditions that permit intra-molecular hybridization, thelabel is typically quenched (or at least altered in its fluorescence) bythe quencher. Under conditions where the MB does not displayintra-molecular hybridization (e.g., when bound to a target nucleicacid, e.g., to a region of an amplicon during amplification), the MBlabel is unquenched. Details regarding standard methods of making andusing MBs are well established in the literature and MBs are availablefrom a number of commercial reagent sources. See also, e.g., Leone etal. (1995) “Molecular beacon probes combined with amplification by NASBAenable homogenous real-time detection of RNA.” Nucleic Acids Res.26:2150-2155; Tyagi and Kramer (1996) “Molecular beacons: probes thatfluoresce upon hybridization” Nature Biotechnology 14:303-308; Blok andKramer (1997) “Amplifiable hybridization probes containing a molecularswitch” Mol Cell Probes 11:187-194; Hsuih et al. (1997) “Novel,ligation-dependent PCR assay for detection of hepatitis C in serum” JClin Microbiol 34:501-507; Kostrikis et al. (1998) “Molecular beacons:spectral genotyping of human alleles” Science 279:1228-1229; Sokol etal. (1998) “Real time detection of DNA:RNA hybridization in livingcells” Proc. Natl. Acad. Sci. U.S.A. 95:11538-11543; Tyagi et al. (1998)“Multicolor molecular beacons for allele discrimination” NatureBiotechnology 16:49-53; Bonnet et al. (1999) “Thermodynamic basis of thechemical specificity of structured DNA probes” Proc. Natl. Acad. Sci,U.S.A. 96:6171-6176; Fang et al. (1999) “Designing a novel molecularbeacon for surface-immobilized DNA hybridization studies” J. Am. Chem.Soc. 121:2921-2922; Marras et al. (1999) “Multiplex detection ofsingle-nucleotide variation using molecular beacons” Genet. Anal.Biomol. Eng. 14:151-156; and Vet et at (1999) “Multiplex detection offour pathogenic retroviruses using molecular beacons” Proc. Natl. Acad.Sci. U.S.A. 96:6394-6399. Additional details regarding MB constructionand use is found in the patent literature, e.g., U.S. Pat. No. 5,925,517(Jul. 20, 1999) to Tyagi et at entitled “Detectably labeled dualconformation oligonucleotide probes, assays and kits;” U.S. Pat. No.6,150,097 to Tyagi et al (Nov. 21, 2000) entitled “Nucleic aciddetection probes having non-FRET fluorescence quenching and kits andassays including such probes” and U.S. Pat. No. 6,037,130 to Tyagi et al(Mar. 14, 2000), entitled “Wavelength-shifting probes and primers andtheir use in assays and kits.”

PCR detection and quantification using dual-labeled fluorogenicoligonucleotide probes, commonly referred to as “TaqMan™” probes, canalso be performed. These probes are composed of short (e.g., 20-25 base)oligodeoxynucleotides that are labeled with two different fluorescentdyes. On the 5′ terminus of each probe is a reporter dye, and on the 3′terminus of each probe a quenching dye is found. The oligonucleotideprobe sequence is complementary to an internal target sequence presentin a PCR amplicon. When the probe is intact, energy transfer occursbetween the two fluorophores and emission from the reporter is quenchedby the quencher by FRET. During the extension phase of PCR, the probe iscleaved by 5′ nuclease activity of the polymerase used in the reaction,thereby releasing the reporter from the oligonucleotide-quencher andproducing an increase in reporter emission intensity. Accordingly,TaqMan™ probes are oligonucleotides that have a label and a quencher,where the label is released during amplification by the exonucleaseaction of the polymerase used in amplification. This provides a realtime measure of amplification during synthesis. A variety of TaqMan™reagents are commercially available, e.g., from Applied Biosystems(Division Headquarters in Foster City, Calif.) as well as from a varietyof specialty vendors such as Biosearch Technologies (e.g., black holequencher probes). Further details regarding dual-label probe strategiescan be found, e.g., in International Publication No. WO 92/02638.

Other similar methods include e.g. fluorescence resonance energytransfer between two adjacently hybridized probes, e.g., using the“LightCycler®” format described in U.S. Pat. No. 6,174,670.

Self-sustained sequence replication can be used to identifypolymorphisms. Self-sustained sequence replication refers to a method ofnucleic acid amplification using target nucleic acid sequences which arereplicated exponentially, in vitro, under substantially isothermalconditions by using three enzymatic activities involved in retroviralreplication: (1) reverse transcriptase, (2) Rnase H, and (3) aDNA-dependent RNA polymerase (Guatelli et al. (1990) Proc Natl Acad SciUSA 87:1874). By mimicking the retroviral strategy of RNA replication bymeans of cDNA intermediates, this reaction accumulates cDNA and RNAcopies of the original target.

Amplified fragment length polymorphisms (AFLP) can also be used toidentify polymorphisms (Vos et al. (1995) Nucl Acids Res 23:4407). Thephrase “amplified fragment length polymorphism” refers to selectedrestriction fragments which are amplified before or after cleavage by arestriction endonuclease. The amplification step allows easier detectionof specific restriction fragments. AFLP allows the detection largenumbers of polymorphic markers and has been used for genetic mapping(Becker et al., (1995) Mol Gen Genet 249:65; and Meksem et al. (1995)Mol Gen Genet 249:74).

Allele-specific hybridization (ASH) can be used to identifypolymorphisms. ASH technology is based on the stable annealing of ashort, single-stranded, oligonucleotide probe to a completelycomplementary single-strand target nucleic acid. Detection may beaccomplished via an isotopic or non-isotopic label attached to theprobe.

For each polymorphism, two or more different ASH probes are designed tohave identical DNA sequences except at the polymorphic nucleotides. Eachprobe will have exact homology with one allele sequence so that therange of probes can distinguish all the known alternative allelesequences. Each probe is hybridized to the target DNA. With appropriateprobe design and hybridization conditions, a single-base mismatchbetween the probe and target DNA will prevent hybridization. In thismanner, only one of the alternative probes will hybridize to a targetsample that is homozygous or homogenous for an allele. Samples that areheterozygous or heterogeneous for two alleles will hybridize to both oftwo alternative probes.

ASH markers are used as dominant markers where the presence or absenceof only one allele is determined from hybridization or lack ofhybridization by only one probe. The alternative allele may be inferredfrom the lack of hybridization. ASH probe and target molecules areoptionally RNA or DNA; the target molecules are any length ofnucleotides beyond the sequence that is complementary to the probe; theprobe is designed to hybridize with either strand of a DNA target; theprobe ranges in size to conform to variously stringent hybridizationconditions, etc.

PCR allows the target sequence for ASH to be amplified from lowconcentrations of nucleic acid in relatively small volumes. Otherwise,the target sequence from genomic DNA is digested with a restrictionendonuclease and size separated by gel electrophoresis. Hybridizationstypically occur with the target sequence bound to the surface of amembrane or, as described in U.S. Pat. No. 5,468,613, the ASH probesequence may be bound to a membrane.

In one embodiment, ASH data are typically obtained by amplifying nucleicacid fragments (amplicons) from genomic DNA using PCR, transferring theamplicon target DNA to a membrane in a dot-blot format, hybridizing alabeled oligonucleotide probe to the amplicon target, and observing thehybridization dots by autoradiography.

Single nucleotide polymorphisms may be detected by differentialmigration patterns of an amplicon comprising the SNP on e.g., anacrylamide gel. However, alternative modes of detection, such ashybridization, e.g., ASH, or RFLP analysis are also appropriate.

5.4.1 Primers and Probes

Primers can readily be designed and synthesized by one of skill in theart for the nucleic acid region of interest. It will be appreciated thatsuitable primers to be used with the invention can be designed using anysuitable method. Primer selection for long-range PCR is described, e.g.,in U.S. Pat. No. 6,898,531, issued May 24, 2005, entitled “Algorithmsfor Selection of Primer Pairs” and U.S. Ser. No. 10/236,480, filed Sep.5, 2002; for short-range PCR, U.S. Ser. No. 10/341,832, filed Jan. 14,2003 provides guidance with respect to primer selection. Also, there arepublicly available programs such as “Oligo” and LASERGENE® available forprimer design. With such available primer selection and design software,the publicly available human genome sequence and the polymorphismlocations provided, one of skill can design primers to amplify the SNPsof the present invention.

In some embodiments, the primers are radiolabelled, or labeled by anysuitable means (e.g., using a non-radioactive fluorescent tag), to allowfor rapid visualization of the different size amplicons following anamplification reaction without any additional labeling step orvisualization step. In some embodiments, the primers are not labeled,and the amplicons are visualized following their size resolution, e.g.,following agarose or acrylamide gel electrophoresis. In someembodiments, ethidium bromide staining of the PCR amplicons followingsize resolution allows visualization of the different size amplicons.

The primers are not limited to generating an amplicon of any particularsize. For example, the primers used to amplify the polymorphisms are notlimited to amplifying the entire region of the RA-CHR9 allele. Theprimers can generate an amplicon of any suitable length. In someembodiments, the amplification produces an amplicon at least 20nucleotides in length, or alternatively, at least 50 nucleotides inlength, or alternatively, at least 100 nucleotides in length, oralternatively, at least 200 nucleotides in length.

Nucleic acid probes for detecting polymorphisms can be cloned and/orsynthesized. It will be appreciated that the precise probe to be usedfor detection of a nucleic acid comprising a SNP can vary. Any suitablelabel can be used with a nucleic acid probe. Detectable labels suitablefor use with nucleic acid probes include, for example, any compositiondetectable by spectroscopic, radioisotopic, photochemical, biochemical,immunochemical, electrical, optical or chemical means. Useful labelsinclude biotin for staining with labeled streptavidin conjugate,magnetic beads, fluorescent dyes, radiolabels, enzymes, and colorimetriclabels. Other labels include ligands which bind to antibodies labeledwith fluorophores, chemiluminescent agents, and enzymes. A probe canalso constitute radiolabelled PCR primers that are used to generate aradiolabelled amplicon. Labeling strategies for labeling nucleic acidsand corresponding detection strategies can be found, e.g., in Haugland(2003) Handbook of Fluorescent Probes and Research Chemicals NinthEdition by Molecular Probes, Inc. (Eugene Oreg.).

A primer or probe is typically at least about 8 nucleotides in length.In one embodiment, a primer or a probe is at least about 10 nucleotidesin length. In a specific embodiment, a primer or a probe is at leastabout 12 nucleotides in length. In another specific embodiment, a primeror probe is at least about 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25nucleotides in length. While the maximal length of a probe can be aslong as the target sequence to be detected, depending on the type ofassay in which it is employed, it is typically less than about 50, 60,65, or 70 nucleotides in length. In the case of a primer, it istypically less than about 30 nucleotides in length. In a specificembodiment, a primer or a probe is within the length of about 18 andabout 28 nucleotides. However, in other embodiments, such as nucleicacid arrays and other embodiments in which probes are affixed to asubstrate, the probes can be longer, such as on the order of 30-70, 75,80, 90, 100, or more nucleotides in length.

For analyzing SNPs, it can be appropriate to use oligonucleotidesspecific for alternative SNP alleles. Such oligonucleotides which detectsingle nucleotide variations in target sequences may be referred to bysuch terms as “allele-specific oligonucleotides,” “allele-specificprobes,” or “allele-specific primers.” The design and use ofallele-specific probes for analyzing polymorphisms is described in,e.g., Mutation Detection A Practical Approach, ed. Cotton et al. OxfordUniversity Press, 1998; Saiki et al., Nature 324, 163-166 (1986);Dattagupta, EP235,726; and Saiki, WO 89/11548.

While the design of each allele-specific primer or probe depends onvariables such as the precise composition of the nucleotide sequencesflanking a SNP position in a target nucleic acid molecule, and thelength of the primer or probe, another factor in the use of primers andprobes is the stringency of the condition under which the hybridizationbetween the probe or primer and the target sequence is performed. Higherstringency conditions utilize buffers with lower ionic strength and/or ahigher reaction temperature, and tend to require a more perfect matchbetween probe/primer and a target sequence in order to form a stableduplex. If the stringency is too high, however, hybridization may notoccur at all. In contrast, lower stringency conditions utilize bufferswith higher ionic strength and/or a lower reaction temperature, andpermit the formation of stable duplexes with more mismatched basesbetween a probe/primer and a target sequence. By way of example and notlimitation, exemplary conditions for high stringency hybridizationconditions using an allele-specific probe are as follows:Prehybridization with a solution containing 5×. standard salinephosphate EDTA (SSPE), 0.5% NaDodSO₄ (SDS) at 55° C., and incubatingprobe with target nucleic acid molecules in the same solution at thesame temperature, followed by washing with a solution containing 2×SSPE,and 0.1% SDS at 55° C. or room temperature. Moderate stringencyhybridization conditions may be used for allele-specific primerextension reactions with a solution containing, e.g., about 50 mM KCl atabout 46° C. Alternatively, the reaction may be carried out at anelevated temperature such as 60° C. In another embodiment, a moderatelystringent hybridization condition suitable for oligonucleotide ligationassay (OLA) reactions wherein two probes are ligated if they arecompletely complementary to the target sequence may utilize a solutionof about 100 mM KCl at a temperature of 46° C.

In a hybridization-based assay, allele-specific probes can be designedthat hybridize to a segment of target DNA from one individual but do nothybridize to the corresponding segment from another individual due tothe presence of different polymorphic forms (e.g., alternative SNPalleles/nucleotides) in the respective DNA segments from the twoindividuals. Hybridization conditions should be sufficiently stringentthat there is a significant detectable difference in hybridizationintensity between alleles, and preferably an essentially binaryresponse, whereby a probe hybridizes to only one of the alleles orsignificantly more strongly to one allele. While a probe may be designedto hybridize to a target sequence that contains a SNP site such that theSNP site aligns anywhere along the sequence of the probe, the probe ispreferably designed to hybridize to a segment of the target sequencesuch that the SNP site aligns with a central position of the probe(e.g., a position within the probe that is at least three nucleotidesfrom either end of the probe). This design of probe generally achievesgood discrimination in hybridization between different allelic forms.

Allele-specific probes are often used in pairs (or, less commonly, insets of 3 or 4, such as if a SNP position is known to have 3 or 4alleles, respectively, or to assay both strands of a nucleic acidmolecule for a target SNP allele), and such pairs may be identicalexcept for a one nucleotide mismatch that represents the allelicvariants at the SNP position. Commonly, one member of a pair perfectlymatches a reference form of a target sequence that has a more common SNPallele (i.e., the allele that is more frequent in the target population)and the other member of the pair perfectly matches a form of the targetsequence that has a less common SNP allele (i.e., the allele that israrer in the target population). In the case of an array, multiple pairsof probes can be immobilized on the same support for simultaneousanalysis of multiple different polymorphisms.

In general, synthetic methods for making oligonucleotides, includingprobes, primers, molecular beacons, PNAs, LNAs (locked nucleic acids),etc., are well known. For example, oligonucleotides can be synthesizedchemically according to the solid phase phosphoramidite triester methoddescribed by Beaucage and Caruthers (1981), Tetrahedron Letts.,22(20):1859-1862, e.g., using a commercially available automatedsynthesizer, e.g., as described in Needham-VanDevanter et al. (1984)Nucleic Acids Res., 12:6159-6168. Oligonucleotides, including modifiedoligonucleotides can also be ordered from a variety of commercialsources known to persons of skill. There are many commercial providersof oligo synthesis services, and thus this is a broadly accessibletechnology. Any nucleic acid can be custom ordered from any of a varietyof commercial sources, such as The Midland Certified Reagent Company(mcrc@oligos.com), The Great American Gene Company (www.genco.com),ExpressGen Inc. (www.expressgen.com), Operon Technologies Inc. (Alameda,Calif.) and many others. Similarly, PNAs can be custom ordered from anyof a variety of sources, such as PeptidoGenic (pkim@ccnet.com), HTIBio-products, inc. (htibio.com), BMA Biomedicals Ltd (U.K.),Bio-Synthesis, Inc., and many others.

5.4.1. Arrays

Array-based detection can be performed to detect polymorphisms.Commercially available arrays, e.g., from Affymetrix (Santa Clara,Calif.) or other manufacturers be used to detect polymorphisms. Reviewsregarding the operation of nucleic acid arrays include Sapolsky et al(1999) “High-throughput polymorphism screening and genotyping withhigh-density oligonucleotide arrays,” Genetic Analysis: BiomolecularEngineering 14:187.192; Lockhart (1998) “Mutant yeast on drugs” NatureMedicine 4:1235-1236; Fodor (1997) “Genes, Chips and the Human Genome.”FASEB Journal 11:A879; Fodor (1997) “Massively Parallel Genomics.”Science 277: 393-395; and Chee et al. (1996) “Accessing GeneticInformation with High-Density DNA Arrays.” Science 274-610-614. In aspecific embodiment, array based detection is the method used for theidentification polymorphisms, due to the inherently high-throughputnature of array based detection.

A variety of probe arrays have been described in the literature and canbe used in the context of the present invention for detection ofpolymorphisms that can be correlated to the phenotypes noted herein. Forexample, DNA probe array chips or larger DNA probe array wafers (fromwhich individual chips would otherwise be obtained by breaking up thewafer) are used in one embodiment of the invention. DNA probe arraywafers generally comprise glass wafers on which high density arrays ofDNA probes (short segments of DNA) have been placed. Each of thesewafers can hold, for example, approximately 60 million DNA probes thatare used to recognize longer sample DNA sequences (e.g., fromindividuals or populations, e.g., that comprise polymorphisms ofinterest). The recognition of sample DNA by the set of DNA probes on theglass wafer takes place through DNA hybridization. When a DNA samplehybridizes with an array of DNA probes, the sample binds to those probesthat are complementary to the sample DNA sequence. By evaluating towhich probes the sample DNA for an individual hybridizes more strongly,it is possible to determine whether a known sequence of nucleic acid ispresent or not in the sample, thereby determining whether a polymorphismfound in the nucleic acid is present. One can also use this approach toperform ASH, by controlling the hybridization conditions to permitsingle nucleotide discrimination, e.g., for SNP identification and forgenotyping a sample for one or more SNPs.

The use of DNA probe arrays to obtain allele information typicallyinvolves the following general steps: design and manufacture of DNAprobe arrays, preparation of the sample, hybridization of sample DNA tothe array, detection of hybridization events and data analysis todetermine sequence. Preferred wafers are manufactured using a processadapted from semiconductor manufacturing to achieve cost effectivenessand high quality, and are available, e.g., from Affymetrix, Inc of SantaClara, Calif.

For example, probe arrays can be manufactured by light-directed chemicalsynthesis processes, which combine solid-phase chemical synthesis withphotolithographic fabrication techniques as employed in thesemiconductor industry. Using a series of photolithographic masks todefine chip exposure sites, followed by specific chemical synthesissteps, the process constructs high-density arrays of oligonucleotides,with each probe in a predefined position in the array. Multiple probearrays can be synthesized simultaneously on a large glass wafer. Thisparallel process enhances reproducibility and helps achieve economies ofscale.

Once fabricated, DNA probe arrays can be used to obtain data regardingpresence and/or expression levels for polymorphisms of interest. The DNAsamples may be tagged with biotin and/or a fluorescent reporter group bystandard biochemical methods. The labeled samples are incubated with anarray, and segments of the samples bind, or hybridize, withcomplementary sequences on the array. The array can be washed and/orstained to produce a hybridization pattern. The array is then scannedand the patterns of hybridization are detected by emission of light fromthe fluorescent reporter groups. Additional details regarding theseprocedures are found in the examples below. Because the identity andposition of each probe on the array is known, the nature of the DNAsequences in the sample applied to the array can be determined. Whenthese arrays are used for genotyping experiments, they can be referredto as genotyping arrays.

The nucleic acid sample to be analyzed is isolated, amplified and,typically, labeled with biotin and/or a fluorescent reporter group. Thelabeled nucleic acid sample is then incubated with the array using afluidics station and hybridization oven. The array can be washed and orstained or counter-stained, as appropriate to the detection method.After hybridization, washing and staining, the array is inserted into ascanner, where patterns of hybridization are detected. The hybridizationdata are collected as light emitted from the fluorescent reporter groupsalready incorporated into the labeled nucleic acid, which is now boundto the probe array. Probes that most clearly match the labeled nucleicacid produce stronger signals than those that have mismatches. Since thesequence and position of each probe on the array are known, bycomplementarity, the identity of the nucleic acid sample applied to theprobe array can be identified.

In one embodiment, two DNA samples may be differentially labeled andhybridized with a single set of the designed genotyping arrays. In thisway two sets of data can be obtained from the same physical arrays.Labels that can be used include, but are not limited to, cychrome,fluorescein, or biotin (later stained with phycoerythrin-streptavidinafter hybridization). Two-color labeling is described in U.S. Pat. No.6,342,355, incorporated herein by reference in its entirety. Each arraymay be scanned such that the signal from both labels is detectedsimultaneously, or may be scanned twice to detect each signalseparately.

Intensity data is collected by the scanner for all the polymorphisms foreach of the individuals that are tested for presence of thepolymorphism. The measured intensities are a measure indicative of theamount of a particular polymorphism present in the sample for a givenindividual (expression level and/or number of copies of the allelepresent in an individual, depending on whether genomic or expressednucleic acids are analyzed). This can be used to determine whether theindividual is homozygous or heterozygous for the polymorphism ofinterest. The intensity data is processed to provide correspondingpolymorphism information for the various intensities.

5.5. Kits

Presented herein are kits for detecting the presence of particularpolymorphisms. Kits useful in diagnosis and prognosis include reagentscomprising, for example, instructions for use and analysis; means forcollecting a tissue or cell sample; nucleic acid probes or primers(e.g., for amplification, reverse transcriptase and detection); labels(e.g., for nucleic acids or proteins); microarrays, gels, membranes orother detection apparati; restriction enzymes (e.g., for RFLP analysis);allele-specific probes; and antisense nucleic acids, any of which may belabeled. In a specific embodiment, the instructions recommend thatpositive and negative controls are run in parallel with test samples. Insome embodiments, the kits comprise one or more control elements, e.g.,oligonucleotides, such as primers and probes.

In specific embodiments, the kits comprise, in one or more containers,one or more reagents employed in the various methods for detecting apolymorphism, such as: (1) reagents for purifying nucleic acids; (2)primers for generating test nucleic acids; (3) dNTPs and/or rNTPs(either premixed or separate), optionally with one or more uniquelylabeled dNTPs and/or rNTPs (e.g., biotinylated or Cy3 or Cy5 taggeddNTPs); (4) post synthesis labeling reagents, such as chemically activederivatives of fluorescent dyes; (5) enzymes, such as reversetranscriptases, DNA polymerases, and the like; (6) various buffermediums, e.g., hybridization and washing buffers; (7) labeled probepurification reagents and components, like spin columns, etc.; and (8)protein purification reagents; (9) signal generation and detectionreagents, e.g., streptavidin-alkaline phosphatase conjugate,chemifluorescent or chemiluminescent substrate, and the like.

In some embodiments, the kits are PCR kits. In one embodiment, the PCRkit includes the following: (a) primers used to amplify a polymorphism;and (b) buffers and enzymes including DNA polymerase.

In some embodiments, the kits are microarray kits. For nucleic acidmicoarray kits, the kits generally comprise probes attached to a solidsupport surface. The probes may be labeled with a detectable label. In aspecific embodiment, the probes are specific for a polymorphism. Themicroarray kits may comprise instructions for performing the assay andmethods for interpreting and analyzing the data resulting from theperformance of the assay. The kits may also comprise hybridizationreagents and/or reagents necessary for detecting a signal produced whena probe hybridizes to a target nucleic acid sequence. Generally, thematerials and reagents for the microarray kits are in one or morecontainers. Each component of the kit is generally in its own a suitablecontainer.

In some embodiments, the kits are microarray kits which do not analyzethe whole genome (i.e., they are not genome arrays). In certainembodiments, the oligonucleotides specific for the haplotypes, SNPs oralleles or other polymorphisms described herein in a microarray arespatially arranged in a manner that facilitates quick analysis ofresults by detecting certain patterns, wherein detection of one patternindicates that a human subject is at increased risk for coronary heartdisease and another pattern indicates that the human is not at increasedrisk for coronary heart disease. In certain embodiments, the kitscomprise one or more control elements e.g., oligonucleotides.

In some embodiments, a kit comprises, in one or more containers, one ormore primers and/or one or more probes for detecting an RA-CHR9 allele.In some embodiments, a kit comprises, in one or more containers, one ormore primers and/or one or more probes for detecting a risk allele in anapproximately 58 kb region extending from about position 22,062,301 toabout position 22,120,389 of human chromosome 9. In certain embodiments,the risk allele is in a region extending from position 22062301+/−5808,3000, 4000, 3000, 2500, 2000, 1101, 1000, 550, 500, 250, 200, 150, 100or 50 nucleotides to position 22120389+/−5808, 5000, 4000, 3000, 2500,2000, 1101, 1000, 550, 500, 250, 200, 150, 100 or 50 nucleotides ofhuman chromosome 9. In a specific embodiment, the kit further comprisesinstructions for detecting an RA-CHR9 allele and evaluating the results.In certain embodiments, the kit further comprises one or more primersand/or one or more probes for detecting a polymorphism identified asbeing associated with an increased likelihood of coronary heart disease,such as those described in International Publication No. WO 2007/006862,WO 2004/03576, WO 2004/035741, and WO 2006/105439; Shiffman et al (2005)Am J Hum Genet 77: 596-605; Shiffman et at (2006) Arteriosclerosis,Thrombosis, and Vascular Biology 26: 1613-1618; Iakoubova et al. (2006)Arteriosclerosis, Thrombosis, and Vascular Biology 26: 2763-2768;Morrison et al. (2007) American Journal of Epidemiology 166: 28-35; Lukeet al. (2007) Arteriosclerosis, Thrombosis, and Vascular Biology 27:2030-2036; Shiffman et al. (2008) Arteriosclerosis, Thrombosis, andVascular Biology 28: 173-179; Iakoubova et al. (2008) J Am Coll Cardiol51: 449-455; Iakoubova et at. (2008) J Am Coll Cardiol 51: 435-443;Shiffman et al. (2008) J Am Coll Cardiol 51:444-448; Bare et al. (2007)Genetics in Medicine 9: 682-689; Helgadottir et alt. (2004) Nat. Genet.36: 233-239; and U.S. Patent Application Publication Nos. 2007/0280917,2006/0019269, 2005/0282855, 2005/0164220, 2005/0113408, 2007/0031847,2006/0228715, 2006/0223093, 2005/0272054, 2007/0072821 and 2005/0112611(each of which are incorporated by herein by reference).

In some embodiments, a kit comprises, in one or more containers, one ormore primers and/or one or more probes for detecting a human chromosome9 haplotype comprising a guanine nucleotide at position 22086055(rs10757274) and/or a guanine nucleotide at position 22105026(rs2383206). In other embodiments, a kit comprises, in one or morecontainers, one or more primers and/or probes for detecting a humanchromosome 9 haplotype comprising a guanine nucleotide at position22086055 (rs10757274) and/or a guanine nucleotide at position 22105026(rs2383206), and at least one, two, three, or more, all or anycombination of the SNPs, insertions and/or deletions recited in Table 7,Table 8, Table 10 or Table 11. In specific embodiments, a kit comprises,in one or more containers, one or more primers and/or probes fordetecting a haplotype in a region extending from position22062301+/−5808, 5000, 4000, 3000, 2500, 2000, 1101, 1000, 550, 500,250, 200, 150, 100 or 50 nucleotides to position 22120389+/−5808, 5000,4000, 3000, 2500, 2000, 1101, 1000, 550, 500, 250, 200, 150, 100 or 50nucleotides of human chromosome 9, wherein the haplotype comprises aguanine nucleotide at position 22086055 (rs10757274) and/or a guaninenucleotide at position 22105026 (rs2383206). In a specific embodiment,the kit further comprises instructions for detecting the haplotype andevaluating the results. In certain embodiments, the kit furthercomprises one or more primers and/or one or more probes for detecting apolymorphism identified as being associated with an increased likelihoodof coronary heart disease, such as those described in InternationalPublication No. WO 2007/006862, WO 2004/03576, WO 2004/035741, and WO2006/105439; Shiffman et al (2005) Am J Hum Genet 77: 596-605; Shiffmanet a (2006) Arteriosclerosis, Thrombosis, and Vascular Biology 26:1613-1618; Iakoubova at al. (2006) Arteriosclerosis, Thrombosis, andVascular Biology 26: 2763-2768; Morrison at al. (2007) American Journalof Epidemiology 166: 28-35; Luke et al. (2007) Arteriosclerosis,Thrombosis, and Vascular Biology 27: 2030-2036; Shiffman et al. (2008)Arteriosclerosis, Thrombosis, and Vascular Biology 28: 173-179;Iakoubova et al. (2008) J Am Coll Cardiol 51: 449-455; Iakoubova at al.(2008) J Am Coll Cardiol 51: 435-443; Shiffman et al. (2008) J Am CollCardiol 51:444-448; Bare et al. (2007) Genetics in Medicine 9: 682-689;Helgadottir et al. (2004) Nat. Genet. 36: 233-239; and U.S. PatentApplication Publication Nos. 2007/0280917, 2006/0019269, 2005/0282855,2005/0164220, 2005/0113408, 2005/0112611, 2007/0031847, 2006/0228715,2006/0223093, 2007/0072821 and 2005/0272054 (each of which areincorporated herein by reference).

In some embodiments, a kit comprises, in one or more containers, one ormore primers and/or one or more probes for detecting a polymorphism witha linkage disequilibrium of between 0.5 to 1, 0.5 to 0.90, or 0.5 to0.80, or 0.5 to 0.75 with a risk allele in an approximately 58 kb regionextending from about position 22,062,301 to about position 22,120,389 ofhuman chromosome 9. In some embodiments, the risk allele is in a regionextending from position 22,062,301+/−5808, 5000, 4000, 3000, 2500, 2000,1101, 1000, 550, 500, 250, 200, 150, 100 or 50 nucleotides to position22,120,389+/−5808, 5000, 4000, 3000, 2500, 2000, 1101, 1000, 550, 500,250, 200, 150, 100 or 50 nucleotides of human chromosome 9. In aspecific embodiment, the kit further comprises instructions fordetecting the polymorphism and evaluating the results. In certainembodiments, the kit further comprises one or more primers and/or one ormore probes for detecting a polymorphism identified as being associatedwith an increased likelihood of coronary heart disease, such as thosedescribed in International Publication No. WO 2007/006862, WO2004/03576, WO 2004/035741, and WO 2006/105439; Shiffman et at (2005) AmJ Hum Genet 77: 596-605; Shiffman et al (2006) Arteriosclerosis,Thrombosis, and Vascular Biology 26: 1613-1618; Iakoubova et al. (2006)Arteriosclerosis, Thrombosis, and Vascular Biology 26: 2763-2768;Morrison et al. (2007) American Journal of Epidemiology 166: 28-35; Lukeet al. (2007) Arteriosclerosis, Thrombosis, and Vascular Biology 27:2030-2036; Shiffman et al. (2008) Arteriosclerosis, Thrombosis, andVascular Biology 28: 173-179; Iakoubova et al. (2008) J Am Coll Cardiol51: 449-455; Iakoubova et al. (2008) J Am Coll Cardiol 51: 435-443;Shiffman et al. (2008) J Am Coll Cardiol 51:444-448; Bare et al. (2007)Genetics in Medicine 9: 682-689; Helgadottir et al. (2004) Nat. Genet.36: 233-239; and U.S. Patent Application Publication Nos. 2007/0280917,2006/0019269, 200510282855, 2005/0164220, 2005/0113408, 2007/0031847,2006/0228715, 2006/0223093, 2007/0072821, 2005/0272054 and 2005/012611(each of which are incorporated by herein by reference).

In some embodiments, a kit comprises, in one or more containers, one ormore primers and/or one or more probes for detecting one, two, three,four or more, all or any combination of the following SNPs, insertions,and/or deletions listed in Table 7, Table 8, Table 10 and/or Table 11.In a specific embodiment, a kit comprises, in one or more containers,one or more primers or probes for detecting the SNP at rs1075724 and/orthe SNP at rs2383206. In another specific embodiment, at kit comprises,in one or more containers, one or more primers and/or probes fordetecting the SNP at rs1333049 and/or the SNP at rs10757278. In aspecific embodiment, the kit further comprises instructions fordetecting the SNPs, insertions and/or deletions and evaluating theresults. In certain embodiments, the kit further comprises one or moreprimers and/or one or more probes for detecting a polymorphismidentified as being associated with an increased likelihood of coronaryheart disease, such as those described in International Publication No.WO 2007/006862, WO 2004/03576, WO 2004/035741, and WO 2006/105439;Shiffman et al (2005) Am J Hum Genet 77: 596-605; Shiffman et at (2006)Arteriosclerosis, Thrombosis, and Vascular Biology 26: 1613-1618;Iakoubova et al. (2006) Arteriosclerosis, Thrombosis, and VascularBiology 26: 2763-2768; Morrison et al. (2007) American Journal ofEpidemiology 166: 28-35; Luke et al. (2007) Arteriosclerosis,Thrombosis, and Vascular Biology 27: 2030-2036; Shiffman et al. (2008)Arteriosclerosis, Thrombosis, and Vascular Biology 28: 173-179;Iakoubova et al. (2008) J Am Coll Cardiol 51: 449-455; Iakoubova et al.(2008) Am Coll Cardiol 51: 435-443; Shiffman et al. (2008) J Am CollCardiol 51:444-448; Bare et al. (2007) Genetics in Medicine 9: 682-689;Helgadottir et al. (2004) Nat. Genet. 36: 233-239; and U.S. PatentApplication Publication Nos. 2007/0280917, 2006/0019269, 2005/0282855,2005/0164220, 2005/013408, 2005/0112611, 2007/0031847, 2006/0228715,2006/0223093, 2007/0072821 and 2005/0272054 (each of which areincorporated by herein by reference).

5.6. Systems

Presented herein are systems comprising a kit or a component(s) of thekits presented herein and a computer program product for use inconjunction with a computer system. In such systems, the computerprogram product can comprise a computer readable storage medium and acomputer program mechanism embedded therein. The computer programmechanism may comprise instructions for evaluating the presence ofparticular polymorphism, in one or a plurality of samples. In someembodiments, the computer program comprises instructions for evaluatingthe presence of one, two, three or more polymorphisms. In someembodiments, the computer program will comprise instructions thatcorrelate the presence or absence of a polymorphism with a predictedphenotype. The system instructions can compare detected information asto allele sequence or expression level with a database that includescorrelations between the alleles and the relevant phenotypes. Thisdatabase can be multidimensional, thereby including higher-orderrelationships between combinations of alleles and the relevantphenotypes. These relationships can be stored in any number of look-uptables, e.g., taking the form of spreadsheets (e.g., Excel™spreadsheets) or databases such as an Access™, SQL™, Oracle™, Paradox™,or similar database. The system includes provisions for inputtingsample-specific information regarding allele detection information,e.g., through an automated or user interface and for comparing thatinformation to the look up tables.

In some embodiments, the system instructions can also include softwarethat accepts diagnostic information associated with any detected alleleinformation, e.g., a diagnosis that a subject with the relevant allelehas a particular phenotype. This software can be heuristic in nature,using such inputted associations to improve the accuracy of the look uptables and/or interpretation of the look up tables by the system. Avariety of such approaches, including neural networks, Markov modeling,and other statistical analysis are described above.

Optionally, system components for interfacing with a user are provided.For example, the systems can include a user viewable display for viewingan output of computer-implemented system instructions, user inputdevices (e.g., keyboards or pointing devices such as a mouse) forinputting user commands and activating the system, etc. Typically, thesystem of interest includes a computer, wherein the variouscomputer-implemented system instructions are embodied in computersoftware, e.g., stored on computer readable media.

Standard desktop applications such as word processing software (e.g.,Microsoft Word™ or Corel WordPerfect™) and database software (e.g.,spreadsheet software such as Microsoft Excel™, Corel Quattro Pro™, ordatabase programs such as Microsoft Access™ or Sequel™, Oracle™,Paradox™) can be adapted to the present invention by inputting acharacter string corresponding to an allele herein, or an associationbetween an allele and a phenotype. For example, the systems can includesoftware having the appropriate character string information, e.g., usedin conjunction with a user interface (e.g., a GUI in a standardoperating system such as a Windows, Macintosh or LINUX system) tomanipulate strings of characters. Specialized sequence alignmentprograms such as BLAST can also be incorporated into the systems of theinvention for alignment of nucleic acids or proteins (or correspondingcharacter strings) e.g., for identifying and relating multiple alleles.

Systems can include a computer with an appropriate database and anallele sequence or correlation of the invention. Software for aligningsequences, as well as data sets entered into the software systemcomprising any of the sequences herein can be a feature of theinvention. The computer can be, e.g., a PC (Intel x86 or Pentiumchip-compatible DOS™, OS2™ WINDOWS™ WINDOWS NT™, WINDOWS95™, WINDOWS98™,WINDOWS2000, WINDOWSME, WINDOWSXP, WINDOWS VISTA or LINUX based machine,a MACINTOSH™, Power PC, or a UNIX based (e.g., SUN™ work station orLINUX based machine) or other commercially common computer which isknown to one of skill. Software for entering and aligning or otherwisemanipulating sequences is available, e.g. BLASTP and BLASTN, or caneasily be constructed by one of skill using a standard programminglanguage such as Visualbasic, Fortran, Basic, Java, or the like.

In some embodiments, the systems include data acquisition modules fordetecting one or more polymorphisms (e.g., one or more array comprisingone or more biomolecular probes, detectors, fluid handlers, or thelike). The biomolecular probes of such a data acquisition module caninclude any that are appropriate for detecting the polymorphism, e.g.,oligonucleotide probes, proteins, aptamers, antibodies, etc. These caninclude sample handlers (e.g., fluid handlers), robotics, microfluidicsystems, nucleic acid or protein purification modules, arrays (e.g.,nucleic acid arrays), detectors, thermocyclers or combinations thereof,e.g., for acquiring samples, diluting or aliquoting samples, purifyingmarker materials (e.g., nucleic acids or proteins), amplifying markernucleic acids, detecting amplified marker nucleic acids, and the like.

For example, automated devices that can be incorporated into the systemsherein have been used to assess a variety of biological phenomena,including, e.g., expression levels of genes in response to selectedstimuli (Service (1998) “Microchips Arrays Put DNA on the Spot” Science282:396-399), high throughput DNA genotyping (Zhang et al. (1999)“Automated and Integrated System for High-Throughput DNA GenotypingDirectly from Blood” Anal. Chem. 71:1138-1145) and many others.Similarly, integrated systems for performing mixing experiments, DNAamplification, DNA sequencing and the like are also available. See,e.g., Service (1998) “Coming Soon: the Pocket DNA Sequencer” Science282: 399-401. A variety of automated system components are available,e.g., from Caliper Technologies (Hopkinton, Mass.), which utilizevarious Zymate systems, which typically include, e.g., robotics andfluid handling modules. Similarly, the common ORCA® robot, which is usedin a variety of laboratory systems, e.g., for microtiter traymanipulation, is also commercially available, e.g., from BeckmanCoulter, Inc. (Fullerton, Calif.). Similarly, commercially availablemicrofluidic systems that can be used as system components in thepresent invention include those from Agilent technologies and theCaliper Technologies. Furthermore, the patent and technical literatureincludes numerous examples of microfluidic systems, including those thatcan interface directly with microwell plates for automated fluidhandling.

Any of a variety of liquid handling and/or array configurations can beused in the systems herein. One common format for use in the systemsherein is a microtiter plate, in which the array or liquid handlerincludes a microtiter tray. Such trays are commercially available andcan be ordered in a variety of well sizes and numbers of wells per tray,as well as with any of a variety of functionalized surfaces for bindingof assay or away components. Common trays include the ubiquitous 96 wellplate, with 384 and 1536 well plates also in common use. Samples can beprocessed in such trays, with all of the processing steps beingperformed in the trays. Samples can also be processed in microfluidicapparatus, or combinations of microtiter and microfluidic apparatus.

In addition to liquid phase arrays, components can be stored in oranalyzed on solid phase arrays. These arrays fix materials in aspatially accessible pattern (e.g., a grid of rows and columns) onto asolid substrate such as a membrane (e.g., nylon or nitrocellulose), apolymer or ceramic surface, a glass or modified silica surface, a metalsurface, or the like. Components can be accessed, e.g., byhybridization, by local rehydration (e.g., using a pipette or otherfluid handling element) and fluidic transfer, or by scraping the arrayor cutting out sites of interest on the array.

The system can also include detection apparatus that is used to detectallele information, using any of the approached noted herein. Forexample, a detector configured to detect real-time PCR products (e.g., alight detector, such as a fluorescence detector) or an array reader canbe incorporated into the system. For example, the detector can beconfigured to detect a light emission from a hybridization oramplification reaction comprising an allele of interest, wherein thelight emission is indicative of the presence or absence of the allele.Optionally, an operable linkage between the detector and a computer thatcomprises the system instructions noted above is provided, allowing forautomatic input of detected allele-specific information to the computer,which can, e.g., store the database information and/or execute thesystem instructions to compare the detected allele specific informationto the look up table.

Probes that are used to generate information detected by the detectorcan also be incorporated within the system, along with any otherhardware or software for using the probes to detect the amplicon. Thesecan include thermocycler elements (e.g., for performing PCR or LCRamplification of the allele to be detected by the probes), arrays uponwhich the probes are arrayed and/or hybridized, or the like. The fluidhandling elements noted above for processing samples, can be used formoving sample materials (e.g., template nucleic acids and/or proteins tobe detected) primers, probes, amplicons, or the like into contact withone another. For example, the system can include a set of probes orprimers configured to detect at least one allele associated with aphenotype. The detector module is configured to detect one or moresignal outputs from the set of probes or primers, or an ampliconproduced from the set of probes or primers, thereby identifying thepresence or absence of the allele.

The sample to be analyzed is optionally part of the system, or can beconsidered separate from it. The sample optionally includes e.g.,genomic DNA, amplified genomic DNA, cDNA, amplified cDNA, RNA, amplifiedRNA, proteins, etc., as noted herein.

Some systems presented herein comprise a kit or one or more componentsof the kits presented herein, a computer having a central processingunit and a memory coupled to the central processing unit. Some systemspresented herein comprise a kit or one or more components of the kitspresented herein, a computer readable medium, a computer having acentral processing unit and a memory coupled to the central processingunit. The memory stores instructions for evaluating the presence ofparticular polymorphisms. In specific embodiments, the memory storesinstructions for evaluating the presence of one, two, three or morepolymorphisms. In some embodiments, the memory comprises instructionsfor transmitting the results of a method presented herein to a remotecomputer and the remote computer includes instructions for evaluatingthe presence of one, two, three or more polymorphisms.

In some embodiments, presented herein is a computer system comprising acomputer readable medium comprising the results of an evaluation for thepresence of particular polymorphisms, as described herein. In someembodiments, a computer system presented herein comprises:

a central processing unit;

a main non-volatile storage unit, for example, a hard disk drive, forstoring software and data, the storage unit controlled by storagecontroller;

a system memory, such as high speed random-access memory (RAM), forstoring system control programs, data and application programs,comprising programs and data loaded from non-volatile storage unit, andmay also include a read-only memory (ROM);

a user interface, comprising one or more input devices (e.g., akeyboard) and display or other output device;

a network interface card for connecting to any wired or wirelesscommunication network (e.g., a wide area network such as the Internet);

an internal bus for interconnecting the aforementioned elements of thesystem; and

a power source to power the aforementioned elements.

Operation of the computer can be controlled primarily by an operatingsystem, which is executed by a central processing unit. The operatingsystem can be stored in the system memory. In addition to the operatingsystem, an implementation system may include: a file system forcontrolling access to the various files and data structures presentedherein; a training data set for use in the construction of one or moredecision rules in accordance with the methods presented herein; a dataanalysis algorithm module for processing training data and constructingdecision rules; one or more decision rules; a profile evaluation modulefor determining whether a polymorphism is present.

The computer may comprise software program modules and data structures.Each of the data structures can comprise any form of a data storagesystem, including, but not limited to, a flat ASCII or binary file, anExcel spreadsheet, a relational database (e.g., SQL), or an on-lineanalytical processing (OLAP) database (e.g., MDX and/or variantsthereof). In some embodiments, such data structures are each in the formof one or more databases that include a hierarchical structure (e.g., astar schema). In some embodiments, such data structures are each in theform of databases that do nor have explicit hierarchy (e.g., dimensiontables that are not hierarchically arranged).

In some embodiments, each of the data structures stored or accessible tothe computer system are single data structures. In other embodiments,such data structures in fact comprise a plurality of data structures(e.g., databases, files, archives) that may or may not all be hosted bythe same computer. For example, in some embodiments, a training data setmay comprise a plurality of Excel spreadsheets that are stored either onthe computer and/or computers that are addressable by the computeracross wide area network. In another example, a training set maycomprise a database that is either stored on the computer or isdistributed across one or more computers that are addressable by thecomputer across a wide area network.

It will be appreciated that many of the modules and data structuresmentioned above can be located on one or more remote computers. Forexample, in some embodiments, web service-type implementations are used.In such embodiments, an evaluation module can reside on a clientcomputer that is in communication with the computer via a network. Insome embodiments, a profile evaluation module can be an interactive webpage.

In some embodiments, a training data set and/or decision rules are on asingle computer and in other embodiments, one or more of such datastructures and modules are hosted by one or more remote computers. Anyarrangement of the data structures and software modules on one or morecomputers is within the scope the systems presented herein so long asthese data structures and software modules are addressable with respectto each other across a network or by other electronic means.

In some embodiments, a digital signal embodied on a carrier wavecomprises data with respect to a method presented herein. In someembodiments, a digital signal embodied on a carrier wave comprises adetermination as to whether a particular polymorphism is present in asample. In some embodiments, a graphical user interface is provided fordetermining whether a polymorphism is present in a sample. The graphicaluser interface may comprise a display field for displaying a resultencoded in a digital signal embodied on a carrier wave received from aremote computer.

6. EXAMPLE Common Allele on Chromosome 9 Associated with Coronary HeartDisease

This example describes the identification of an approximately 58 kbregion of human chromosome 9 associated with an increased risk ofcoronary heart disease.

Coronary heart disease (CHD) is the single greatest cause of deathworldwide (1, 2). Although CHD is highly heritable, the DNA sequencevariations that confer cardiovascular risk remain largely unknown. Toidentify sequence variants associated with CHD, we undertook agenome-wide association study using 100,000 single nucleotidepolymorphisms (SNPs). To minimize false positive associations withoutunduly sacrificing statistical power, the study design comprised threesequential case-control comparisons performed at a nominal significancethreshold of P<0.025 (FIG. 1). For the initial genome-wide scan, casesand controls were Caucasian men and women from Ottawa, Canada whoparticipated in the Ottawa Heart Study (OHS). Cases had severe,premature CHD with a documented onset before the age of 60 years andculminating in coronary artery revascularization (Table 1). To limitconfounding by factors that strongly predispose to premature CHD,individuals with diabetes or plasma cholesterol levels consistent withmonogenic hypercholesterolemia (>280 mg/dL) were excluded. Controls werehealthy Caucasian men (>65 y) and women (>70 y) from Ottawa who had nosymptoms or history of CHD (Table 1).

Custom oligonucleotide arrays (3) were used to assay 100,000 SNPsarranged at approximately 30 kb intervals throughout the genome in 322cases and 312 controls (OHS-1). Of these, 9,636 SNPs deviated stronglyfrom Hardy-Weinberg equilibrium (P<0.001) or did not meetquality-control criteria (3) and 17,500 were sub-polymorphic (minorallele frequency <1%) in the sample. The remaining 72,864 SNPs wereentered into the analysis and 2,586 were associated with CHD at anominal significance threshold of 0.025 (Table 2). These 2,586 SNPs weregenotyped in an independent sample of 311 cases and 326 controls, fromOttawa (OHS-2) using the same criteria as OHS-1 (Table 1). Of these, 50were associated with CHD at a nominal significance threshold of 0.025,with the same direction of effect (Table 2).

To limit attrition of true positive associations due to inadequatestatistical power, the third case-control comparison was performed in amuch larger prospective study of CHD risk, the Atherosclerosis Risk inCommunities (ARIC) study, which enrolled and followed 11,478 Caucasians(4). Only two of the 50 SNPs identified in the Ottawa cohorts weresignificantly associated with incident CHD in the ARIC population (Table2). These two SNPs, rs10757274 and rs2383206, were located within 20 kbof each other on chromosome 9 and were in strong linkage disequilibrium(r2=0.89).

To validate the association between rs10757274 and rs2383206 and CHD,both SNPs were assayed in three additional independent cohorts: theCopenhagen City Heart Study (CCHS), a prospective study of ischemicheart disease in 10,578 Danish men and women (5); the Dallas Heart Study(DHS), a population-based probability sample of Dallas County residents(6); and a third sample of 647 cases and 847 controls from the OttawaHeart Study population (OHS-3). In the CCHS, cases were participants whoexperienced an ischemic cardiovascular event during the 20 yr follow-upperiod while controls were those who did not develop CHD over the sametime interval. In the DHS, cases and controls were defined usingelectron-beam computer tomography to measure coronary artery calcium, anindex of coronary atherosclerosis (7). In OHS-3, cases had documentedCHD before the age of 55 (men) or 65 (women) years, whereas controlswere men aged >65 and women aged >70 years who did not have symptoms ofCHD (Table 1). In all three validation studies, both SNPs weresignificantly associated with CHD (Table 3).

The magnitude of CHD risk associated with the risk allele was determinedby Cox proportional-hazards modeling in the ARIC and CCHS cohorts. Thehazard ratios associated with the risk alleles were comparable in thetwo populations, and indicated a graded increase in risk fromnoncarriers to heterozygotes to homozygotes (Table 4). The two SNPs(rs10757274 and rs2383206) define an allele that was associated with a˜15-20% increase in risk in the 50% of individuals who were heterozygousfor the allele and a ˜30-40% increase in CHD in the 25% of Caucasianswho were homozygous for the allele. The population attributable riskassociated with the risk allele was 12.5-15% in the ARIC population and10-13% in the CCHS cohort.

Without being bound by theory, it is possible that the associationbetween the risk allele defined by rs10757274 and rs2383206 increasesthe development of atherosclerotic plaques, promotes thrombogenesis, orincreases the tendency of atherosclerotic plaques to rupture. Thefinding that the risk allele was associated with coronary arterycalcification in the DHS and with severe premature atherosclerosis inOHS-1 indicates that it promotes CHD by increasing the atheroscleroticburden. The risk allele was not associated with any of the major riskfactors for atherosclerosis in ARIC or CCHS (Tables 5 and 6), and theassociation remained significant in models that considered multiplepossible confounding covariates (including age, gender, plasma lipidlevels, blood pressure, diabetes, and plasma C-reactive protein levels,see Table 3). These analyses indicates that the effect of the chromosome9 risk allele on CHD was not mediated by any of the established riskfactors for cardiovascular disease.

To fine-map the locus associated with CHD, we assayed SNPs spaced at ˜5kb intervals across the region extending 175 kb upstream and downstreamof rs10757274 and rs2383206 in 500 cases and 500 controls from OHS-2 andOHS-3. Eight additional SNPs at the locus spanning a 58 kb region(extending from 22,062,301 to 22,120,389) were significantly associatedwith CHD (FIG. 2). All eight were in strong linkage disequilibrium witheach other and with rs10757274 and rs2383206. The region demarcated bythese SNPs was flanked on both sides by ˜50 kb regions in which none ofthe 30 SNPs tested were associated with CHD. Two of 65 SNPs in the 350kb region surrounding the 58 kb risk locus were associated with CHD atthe nominal significance threshold, but neither was in strong linkagedisequilibrium with rs10757274 and rs2383206. These data indicate thatthe risk allele comprises a single haplotype that spans ˜58 kb.

Inspection of the UCSC Genome Browser and BLAST searches against theNCBI nr nucleotide sequence database revealed no annotated genes ormicroRNAs within the 58 kb interval. A number of spliced ESTs map withinthe interval, but none contain open reading frames that extend more thana few amino acids. Resequencing of the 58 kb interval in two homozygotesfor the risk allele and one homozygote for the reference allele revealed66 polymorphisms (SNPs plus small insertions or deletions), of which 35were specific to the risk allele (Table 7). Only one of these variants,a copy number variation in a run of 9 consecutive CAT repeats (extendingfrom nucleotide 22110787 to 22110814, NCBI build 36.1) mapped to aspliced transcript (B1765545) that appears to be part of a largenoncoding RNA of unknown function (8). PCR amplification of cDNAsconfirmed expression of the transcript in placenta and transformedlymphocytes. Variation in the expression or function of this transcriptmay be associated with risk of CHD.

Alternatively, the risk allele may alter a regulatory element thataffects the expression of a gene located outside of the 58 kb interval.Cross-species sequence alignments revealed several conserved segmentswithin the 58 kb interval that may contain such regulatory elements. Itis also possible that the risk allele extends beyond the 58 kb intervaldefined in this study, that the functional sequence variants that conferrisk of CHD are located outside of the interval. Resequencing the codingregions of the two genes most proximal to the risk locus, CDKN2A andCDKN2B revealed only a single variant (A 158 in CDKN2A) that was presentin 6 of the 96 individuals examined and is thus unlikely to explain theCHD risk associated with the locus. The localization of the risk locusto a region devoid of known genes implicates a previously unrecognizedgene or regulatory element that can substantially affect CHDindependently of established risk factors.

Comparison of the Yoruba and CEPH data from the HapMap revealed strikingethnic differences in allele frequencies in the risk interval (Table 8).Of the 10 alleles that were significantly associated with CHD in whites,3 were virtually absent from the Yoruba population, and 6 others muchless common. Both rs10757274 and rs2383206 were present at appreciablefrequencies among African-Americans in ARIC and DHS, but neither SNP wasassociated with CHD in either population (Table 9). The apparent ethnicdifferences in association between these SNPs and CHD in ARIC mayreflect differences in statistical power in ARIC, but cannot explain theethnic differences observed in DHS, where African-Americans are thelargest group, indicating that the functional sequence variantsassociated with the risk allele in whites are less common inAfrican-Americans. This notion is consistent with our finding that thefrequencies of several alleles associated with CHD risk factors differwidely among ethnic groups (9-11).

The results of this study illustrate both the perils and the promise ofwhole-genome association. The initial scan and the first replicatescreen both generated substantially more SNPs that achieved thepre-specified significance threshold than would be predicted by chancealone, as indicated by permutation testing (Table 2). Yet only two ofthese SNPs (comprising one allele) survived further replication, despitethe use of a large sample (i.e., ARIC) with high statistical power. Thisfinding highlights the necessity for adequate replication to protectagainst artifacts that may occur due to population stratification,multiple testing, or other factors to which whole-genome associationstudies are particularly susceptible. The consistent replication of thechromosome 9 risk allele in six independent study samples indicates thatthe approach can be productively applied to conditions as complex asCHD, which is known to be influenced by a variety of environmental andgenetic factors (12). Furthermore, analysis of 50 randomly selectedregions of 500 kb each indicated that the 72,864 informative SNPs usedin the initial scan provided 30-40% of the power that would be obtainedby assaying all Phase II Hapmap SNPs.

6.1. Materials and Methods

The Ottawa Heart Study. The Ottawa Heart Study is an ongoing,hospital-based study of coronary heart disease at the Ottawa HeartInstitute in Ottawa, Canada. The study was approved by the InstitutionalReview Board at the University of Ottawa Heart Institute and allparticipants provided written informed consent. All patients at theInstitute who undergo coronary artery bypass grafting, coronary arteryangiography, or care for acute myocardial infarction are invited toparticipate in the study. Three independent samples (OHS-1, OHS-2, andOHS-3) were ascertained serially for this study. Caucasian men and womenaged <60 y with advanced disease requiring coronary artery bypassgrafting or percutaneous coronary intervention who did not have ahistory of diabetes mellitus or severe dyslipidemia, suggestive of amonogenic lipid disorder (TC>280 mg/dl/7.0 mmol/L) were included in theinitial genome-wide scan (OHS-1) Subsequently, a second sample ofindividuals was recruited using the same clinical criteria (OHS-2). Themean age of onset of CAD in these individuals was 47.8+7.5 (SD) years.Once recruitment for OHS-1 and OHS-2 was completed, individuals withdocumented CHD before the age of 55 (men) or 65 (women) were recruitedfor OHS-3. Healthy elderly controls (men >65 y, women >70 y) wererecruited via an extensive newspaper and television advertising campaignin the Ottawa community. Controls were carefully interviewed by aphysician or nurse to ascertain that they were free of symptoms ofpossible ischemic arterial disease and had no past history ofcardiovascular symptoms, a positive stress test, coronary angiographydemonstrating stenosis (>50%) in any artery or clinical cardiovascularevents. Individuals with the same ethnic background as the cases(Caucasian) were included in this study. The mean age of the controlsubjects was 74.9+4.8 years. Controls for OHS-1, OHS-2, and OHS-3 werecollected sequentially as described for cases.

The Atherosclerosis Risk in Communities Study (ARIC). The ARIC studycomprised men and women aged 45 to 64 years who were randomly selectedfrom four communities (Jackson, Miss.; Minneapolis Minn.; ForsythCounty, N.C.: and Washington County, Md.). The protocol for the studywas approved by the institutional review boards of all centers, and allparticipants provided written informed consent that included consent forgenetic studies. Race or ethnic group was determined byself-identification; participants described themselves as black or whitein response to a questionnaire on which the available categories were“black”, “white”, “Indian”, or “Asian”. Plasma lipids, glucose, insulinand lipoproteins were assayed in the ARIC central lipid laboratory withcommercial reagents, as previously described (13-15). Cases had adocumented CHD event (defined as myocardial Infarction, coronary arteryrevascularization, or coronary death) during the 15 yr follow-up periodof the study; individuals with prevalent disease at the baseline visitwere excluded. Controls were individuals who did not develop incidentCHD. The study sample delineated by these criteria provides >90% powerto detect common alleles (minor allele frequency >0.1) that differ infrequency by 0.06 or more between cases and controls.

The Copenhagen City Heart Study (CCHS). The Copenhagen City Heart Studypopulation was randomly drawn from the Copenhagen Population Register inJanuary 1976 (16). The study was approved by the Danish ethics committeefor the City of Copenhagen and Frederiksberg and informed consent wasprovided according to the Declaration of Helsinki. The sample was drawnfrom a population of approximately 90 000 inhabitants 20 years and olderliving within 10 wards surrounding Rigshospitalet, the NationalUniversity Hospital of Copenhagen. A second examination was performed 5years later (1981-1983), and a third examination was performed after 15years (1991-1994), at which time blood samples were obtained from 9,259individuals for isolation of DNA. A self-administered questionnaire wasused to obtain information regarding familial history, education andsocio-economic status, and smoking and drinking habits. Plasmacholesterol and triglyceride levels were determined enzymatically usingcommercial reagents, and HDL-C was determined after removal ofapoB-containing lipoproteins by precipitation with phosphotungstic acidand magnesium. Participants were white and of Danish descent.

The Dallas Heart Study. The Dallas Heart Study is a multi-ethnic,population-based probability sample of Dallas County residents. Thestudy was approved by the Institutional Review Board at the Universityof Texas Southwestern Medical Center and included three phases: anin-home interview, an in-home phlebotomy visit, and a clinic visitduring which a variety of imaging examinations were performed, Caucasianmen and women who underwent electron-beam computer tomography to assesscoronary artery calcification were eligible for the present study. Eachindividual underwent two consecutive scans. The distribution of CACscores is extremely skewed and inter-scan variability is high for scoresbelow 10 Agatston units (17), therefore we excluded individuals with CACscores between 2 and 10 units and divided the population into controls(CAC scores of ≦2 units, n=575) and cases (those with CAC scores ≧10units, n=166), as previously described (5).

Genotyping: Chip-Based Oligonucleotide Hybridization:SNP Selection.

Using NCBI Build 34, the genome was partitioned into blocks of 13 kb,and one SNP was selected from each block. Using a whole-genomemulti-ethnic haplotype map (18), we preferentially chose commonhaplotype defining SNPs, then common SNPs, then rare SNPs. Where nopreviously characterized SNPs were available, we chose validated SNPsfrom dbSNP. Ties were broken so as to minimize variation in inter-SNPspacing. This yielded roughly 200,000 SNPs, of which 70% were haplotypedefining, and another 10% were common, 4% were rare, and 16% were fromdbSNP. Every other SNP was selected, yielding a set of ˜100,000. Usingperformance data for these assays on another array design to identifyfailing assays, we selected replacement SNPs from the multi-ethnic mapto fill the largest gaps.

Genotyping. Genotyping was performed by Perlegen Sciences using customhigh-density oligonucleotide arrays. Each SNP was interrogated by 24different 25mer oligonucleotide probes synthesized on a glass substrate.The 24 features comprise four sets of six features interrogating bothreference and alternate alleles on forward and reference strands. Eachallele and strand is represented by oligonucleotides with the variantnucleotides a five offset positions: 22, 21, 0, 1 and 2, (where thenumber indicates the position of the SNP within the 25mer, with 0 beingthe 13th base). At offset 0, a quartet was tiled, which includes theperfect match to reference and alternate SNP alleles and the tworemaining nucleotides as mismatch probes. When possible, the mismatchfeatures were selected to match a purine nucleotide substitution with apurine nucleotide and a pyrimidine nucleotide with a pyrimidinenucleotide.

The reliability of the intensity measurements of each SNP was assessedusing two methods. One metric, “conformance”, indicates the presence ofspecific target DNA for that SNP. The other metric, “signal tobackground ratio”, measures the relative amounts of specific andnonspecific binding. SNPs that failed to meet specified cutoffs on bothmetrics were discarded. Conformance was computed independently for thetwo allele feature sets, and a maximum was taken of the two values.Conformance of a given allele is defined as the fraction of feature setsfor which the perfect-match feature is brighter than the correspondingmismatch feature. SNP measurements having conformance scores <0.9 werediscarded. The signal to background ratio was calculated from intensitymeasurements for both alleles, as the root mean square of trimmed meanintensities for the perfect-match features for each allele, divided bythe corresponding value for the mismatch features. SNP measurementshaving signal/background <1.5 were discarded.

Calling Algorithm. Individual genotypes for a SNP were determined byclustering measurements from multiple scans in the two-dimensional spacedefined by background-adjusted trimmed mean intensities of theperfect-match features for each allele. A K-means algorithm was used toassign measurements to clusters representing distinct diploid genotypes.The average call rate was 98.54 percent.

Mass Spectrometry. The 50 sequence variants identified in thegenome-wide scan were assayed in the ARIC population by massspectrometry using the Sequenom MassARRAY system (Sequenom, Inc.; SanDiego, Calif.).

Fluorogenic 5′-nucleotidase assays for rs10757274 and rs2383206 weredeveloped with the use of the TaqMan assay system (Applied Biosystems).The assays were performed on a 7900HT Fast Real-Time PCR instrument withprobes and reagents purchased from Applied Biosystems.

DNA sequencing. The 58 kb interval between rs12555547 and rs10965244 wassequenced in two individuals homozygous for the risk allele, and in oneindividual homozygous for the wild-type allele as described (19). Thecoding region and flanking intronic sequences of CDKN2A and CDKN2B weresequenced in 96 arbitrarily selected Caucasian men. All sequencevariants identified were verified by manual inspection of thechromatograms and missense changes were confirmed by an independentresequencing reaction.

Reverse transcription and PCR Amplification of cDNAs. RNA was isolatedfrom human, placenta, and EBV-transformed lymphocytes by a modifiedphenol-chloroform extraction (TRIZOL reagent, Invitrogen Corporation,Carlsbad, Calif.), and reverse transcribed (SuperScript III First-strandsynthesis system, Invitrogen). Aliquots of cDNA were amplified usingprimers directed against spliced ESTs CN277071, BX100299 and DQ485453.

Statistical Analysis. For the genome-wide scan, allelic associationswere evaluated for each SNP using chi-square tests on 2×2 contingencytables with no adjustments. Calculations were performed independently atPerlegen Sciences and at UT Southwestern and essentially identicalresults were obtained. To determine the empirical P-values we randomizedthe sample case/control status 1,000 times, and calculated allelicassociations on each permuted dataset. SNPs that were significantlyassociated with CHD in the genome-wide scan were assayed in a second setof cases and controls and analyzed using Chi-square tests andpermutation testing. For the remaining data sets, case-controldifferences in allele frequencies of rs10757274 and rs2383206 wereevaluated using chi-square tests on 3×2 contingency tables. Populationattributable risk was calculated using the formula PAR=I(T)−I(0), whereI(T) is the total disease incidence in the population, and I(0) is thedisease incidence in unexposed individuals.

TABLE 1 Clinical Characteristics of Participants in the Ottawa HeartStudy. Early CHD Cases Healthy Controls OHS-1* n 322 312 Men (%) 90 55Controls: Age at Recruitment N/A 69.0 ± 6.1 Cases: Age at Diagnosis 48.5± 7.2 N/A MI (%) 62 0 PCI (%) 57 0 CABG (%) 74 0 Cholesterol (mg/dL)218.9 ± 34.4 216.9 ± 36.7 Triglycerides (mg/dL)  215.2 ± 124.9 128.4 ±71.7 LDL-C (mg/dL) 137.7 ± 30.9 135.3 ± 32.1 HDL-C (mg/dL)  40.2 ± 15.1 56.5 ± 15.1 Hypertension (%) 37 33 Cigarette Smoking, ever (%) 60 36BMI (kg/m²) 28.5 ± 4.1 28.0 ± 6.3 OHS-2^(†) n 311 326 Men (%) 80 74%Controls: Age at Recruitment N/A 70.6 ± 6.4 Cases: Age at Diagnosis 47.5± 7.2 N/A MI (%) 65 0 PCI (%) 53 0 CABG (%) 59 0 Cholesterol (mg/dL)226.6 ± 39.1 220.8 ± 37.1 Triglycerides (mg/dL)  225.9 ± 162.1  138.2 ±101.9 LDL-C (mg/dL) 144.2 ± 36.3 141.5 ± 31.7 HDL-C (mg/dL)  41.0 ± 13.1 53.0 ± 18.2 Hypertension (%) 44 23 Cigarette smoking, ever (%) 55 16BMI (kg/m²) 28.8 ± 4.7 26.1 ± 3.7 OHS-3^(‡) n 647 847 Men (%) 76 61Controls: Age at recruitment N/A 72.5 ± 6.0 Cases: Age at diagnosis 49.6± 8.3 N/A MI (%) 66 0 PCI (%) 60 0 CABG (%) 58 0 Cholesterol (mg/dL)231.6 ± 39.8 223.5 ± 40.6 Triglycerides (mg/dL)  194.0 ± 129.3 128.5 ±88.6 LDL-C (mg/dL) 148.1 ± 35.9 141.9 ± 33.2 HDL-C (mg/dL)  45.6 ± 13.5 55.3 ± 17.0 Hypertension (%) 43 32 Cigarette smoking, ever (%) 67 44BMI (kg/m²) 28.6 ± 4.9 26.4 ± 4.0 Lipid values, ages and BMI areexpressed as means ± SD. *Lipid values prior to treatment with lipidmodifying agents; n = 277 cases, n = 285 controls. †Lipid values priorto treatment with lipid modifying agents; n = 195 cases, n = 284controls ‡Clinical data for 580 cases and 822 controls. Lipid valuesprior to treatment with lipid modifying agents; n = 249 cases, n = 418controls. MI, myocardial infarction; PCI, percutaneous coronaryintervention; CABG, coronary artery bypass grafting; LDL-C, low densitylipoprotein-cholesterol; HDL-C, high density lipoprotein-cholesterol;BMI, body mass index

TABLE 2 SNPs associated with CHD in OHS-1, OHS-2 and ARIC. The P-valuesfor the number of SNPs observed relative to the number expected werederived from 1000 permutations in which case-control status wasrandomized. SNPs in the ARIC cohort were tested for association withincident CHD. Individuals with prevalent CHD at baseline were excluded.SNPs with SNPs with SNPs P < 0.025 P < 0.025 Observed- Assayed ObservedExpected Expected Cohort (n) (n) (n) (n) P OHS-1 72,864 2,586 2,066 520<0.001 OHS-2 2,083 50 26 24 <0.001 ARIC 50 2 0 2 NA OHS, Ottawa HeartStudy; ARIC, Atherosclerosis Risk in Communities Study; SNP, singlenucleotide polymorphism.

TABLE 3 Association between SNPs rs10757274 and rs2383206 and CHD.Values are numbers of individuals in each genotype group. P-values werecalculated by Chi-Square tests on allele counts. OHS, Ottawa HeartStudy, ARIC, Atherosclerosis Risk in Communities Study; CCHS, CopenhagenCity Heart Study, DHS, Dallas Heart Study, SNP, single nucleotidepolymorphism. rs10757274 rs2383206 Controls Cases Controls Cases CohortAA AG GG AA AG GG x²-P HW-P AA AG GG AA AG GG x²-P HW-P OHS-1 85 149 7849 148 125 3.7 × 10⁻⁶ 0.08 77 147 88 45 140 137 6.7 × 10⁻⁶ 0.19 OHS-2 85161 80 56 140 108 0.0009 0.4 80 160 86 50 141 113 0.0008 0.34 ARIC 20633822 1858 230 525 282 0.004 0.11 2140 4161 2231 230 600 324 0.0007 0.21CCHS 2752 4543 1758 393 792 340 0.0004 0.56 2489 4583 1981 372 782 3710.016 0.58 DHS 147 258 122 27 85 42 0.025 0.99 131 258 138 24 84 460.045 0.95 OHS-3 228 418 201 121 333 193 0.0003 0.96 197 416 229 115 327209 0.011 0.98

TABLE 4 Risk of CHD as a function of rs10757274 and rs2383206 in theAtherosclerosis Risk in Communities Study and the Copenhagen City HeartStudy. Atherosclerosis Risk in Communities Study Copenhagen City HeartStudy Number of Events Number of Events n (%) Observed Expected¹Incidence² Hazard Ratio n (%) Observed Expected Incidence Hazard Ratiors10757274 AA 2,293 (26)  255³ 295 79 (70-89)  1 3,145 (30)  393⁵ 473 61(55-68) 1 AG 4,347 (50) 564 553 93 (86-101) 1.18 (1.02-1.37) 5,335 (50)792 755 73 (68-79) 1.26 (1.12-1.42) GG 2,140 (24) 298 269 101 (90-114) 1.29 (1.09-1.52) 2,098 (20) 340 296 80 (72-89) 1.38 (1.19-1.60)rs2383206 AA 2,370 (25)  259⁴ 310 78 (69-88)  1 2,861 (27)  372⁶ 425 64(58-71) 1 AG 4,761 (49) 643 610 97 (90-105) 1.26 (1.09-1.46) 5,365 (51)782 772 72 (67-77) 1.16 (1.02-1.31) GG 2,555 (26) 345 327 97 (88-108)1.26 (1.07-1.48) 2,352 (22) 371 327 78 (71-87) 1.29 (1.12-1.50) ¹Basedon the Log-rank test. ²Incidence rate measured in number of events per10,000 person years of follow-up. ³p < 0.0111 ⁴p < 0.0041. ⁵p < 0.00001.⁶p < 0.0

TABLE 5 Clinical and laboratory characteristics of participants in theARIC Study. rs10757274 rs2383206 AA AG GG P-value AA AG GG P-value n2,293 4,347 2,140 — 2,370 4,761 2,555 — Men/Women 1,057/1,2361,972/2,375 958/1,182 0.67 1,106/1,264 2,160/2,601 1,152/1,403 0.48 Age(years) 54 ± 6 54 ± 6 54 ± 6 0.76 54 ± 6 54 ± 6 54 ± 6 0.09 BMI (kg/m²)27.2 ± 5.0 26.9 ± 4.8 26.8 ± 4.7 0.004 27.2 ± 5.0 26.9 ± 4.8 26.9 ± 4.80.01 Systolic BP (mm/Hg) 119 ± 17 118 ± 17 118 ± 17 0.44 119 ± 17 118 ±17 118 ± 17 0.53 Diastolic BP (mm/Hg)  72 ± 10  72 ± 10  71 ± 10 0.28 72 ± 10  71 ± 10  72 ± 10 0.06 Glucose (mg/dL) 106 ± 33 105 ± 32 104 ±25 0.07 105 ± 30 105 ± 32 104 ± 26 0.29 Cholesterol (mg/dL) 215 ± 39 215± 41 213 ± 40 0.16 214 ± 39 215 ± 41 213 ± 40 0.17 Triglyceride (mg/dL)116 (82-167) 112 (80-160) 112 (80-162) 0.16 115 (82-166) 112 (81-160)113 (81-162) 0.28 LDL-C (mg/dL) 137 ± 37 137 ± 38 135 ± 37 0.23 137 ± 37137 ± 39 136 ± 37 0.27 HDL-C (mg/dL)  51 ± 17  51 ± 17  51 ± 16 0.56  51± 17  51 ± 17  51 ± 16 0.16 Values are means ± standard deviations,except for triglycerides which are medians (interquartile ranges).P-values were calculated using ANOVA. Variables with highly skeweddistributions (triglycerides) were log transformed prior to analysis.BMI, body mass index; BP, blood pressure; LDL-C, low densitylipoprotein-cholesterol; HDL-C, high density lipoprotein-cholesterol. Inaddition to the above analyses, both SNPs remained significant in modelsthat included (and excluded) the following co-variates: age, gender,plasma lipid levels, hypertension status, blood pressure levels,diabetes status, glucose and insulin levels, measures of body size(including waist circumference and BMI), measures of inflammation(including CRP, sICAM and sVCAM), environmental exposures includingsmoking, diet (e.g. saturated fat intake and Key's score) and physicalactivity (work and recreational).

TABLE 6 Clinical and laboratory characteristics of participants in theCopenhagen City Heart Study. rs10757274 rs2383206 AA AG GG P-value AA AGGG P-value n 3,145 5,335 2,098 — 2,861 5,365 2,352 — Men/Women1,358/1,787 2,401/2,934 952/1146 0.18 1,233/1,628 2,395/2,9701,083/1,269 0.10 Age (years)  58 ± 15  57 ± 15  57 ± 15 0.29  58 ± 12 58 ± 15  57 ± 15 0.10 BMI (kg/m²) 26 ± 4 26 ± 4 26 ± 4 0.57 25 ± 4 26 ±4 26 ± 4 0.24 Systolic BP (mm/Hg) 139 ± 23 138 ± 22 139 ± 23 0.59 139 ±23 139 ± 22 139 ± 23 0.96 Diastolic BP (mm/Hg)  84 ± 12  84 ± 12  84 ±12 0.38  84 ± 12  84 ± 12  84 ± 12 0.22 Glucose (mg/dL) 104 ± 34 105 ±36 105 ± 34 0.35 105 ± 35 105 ± 35 105 ± 34 0.60 Cholesterol (mg/dL) 237± 50 238 ± 49 236 ± 52 0.40 237 ± 50 238 ± 49 236 ± 52 0.29 Triglyceride(mg/dL) 135 (96-202) 133 (96-196) 137(96-194) 0.98 135 (96-202)136(96-196) 137 (96-194) 0.88 LDL-C (mg/dL) 161 ± 48 162 ± 47 161 ± 470.43 161 ± 48 162 ± 47 161 ± 47 0.40 HDL-C (mg/dL)  61 ± 19  61 ± 19  60± 19 0.64  61 ± 19  61 ± 19  60 ± 19 0.41 Values are means ± standarddeviations, except for triglycerides which are medians (interquartileranges). P-values were calculated using ANOVA. Variables with highlyskewed distributions (triglycerides) were log transformed prior toanalysis. BMI, body mass index; BP, blood pressure; LDL-C, low densitylipoprotein-cholesterol; HDL-C, high density lipoprotein-cholesterol

TABLE 7 Sequence variations identified in the 58 kd risk interval onchromosome 9 in two individuals homozygous for the risk allele and onehomozygous for the alternate allele. Referent Allele Risk GenotypeGenotype Position Type Major Minor 1 2 1 22062264 SNP A G G/G G/G A/A22062301 SNP G C C/C C/C G/G 22062638 SNP G A A/A A/A G/G 22062719 SNP AG G/G G/G A/A 22063996 SNP T G G/G G/G N/N 22067543 SNP C T T/T T/T N/N22071397 SNP G T T/T T/T G/G 22071850 SNP C T T/T T/T C/C 22072375 SNP AC A/C A/A A/A 22073209 SNP C T C/C C/T N/N 22073400 SNP A T A/T A/A N/N22073404 SNP C T T/T T/T N/N 22074310 SNP C T T/T T/T N/N 22075598 SNP TC C/C C/C N/N 22077473 SNP T C C/C C/C C/T 22078090 SNP A T T/T T/T A/A22078094 SNP A G G/G G/G A/A 22078260 SNP C T T/T T/T C/C 22078465 DELCA — —/— —/— CA/CA 22089568 SNP C A A/A A/A N/N 22089755 INS — A A/A A/A—/— 22090176 SNP G C C/C C/C G/G 22091702 SNP T C C/C C/C T/T 22092165SNP C T T/T T/T C/C 22092437 SNP G A G/G G/G A/G 22093183 SNP G T T/TT/T G/G 22093341 SNP T G G/G G/G T/T 22093813 SNP A G G/G G/G A/A22095927 SNP T C C/C C/C T/T 22096225 SNP G A A/A A/A N/N 22096271 SNP AG G/G G/G N/N 22096400 SNP G A A/G G/G N/N 22096731 SNP T A A/A A/A T/T22097238 SNP A T A/A A/A A/T 22100131 SNP T C C/C C/C T/T 22101587 INS —TTGAT TTGAT/ TTGAT/ —/— TTGAT TTGAT 22102241 SNP A C C/C C/C A/A22102427 SNP A G G/G G/G A/A 22102599 SNP T C C/C C/C N/N 22104469 SNP GC C/C C/C G/G 22104495 SNP A G G/G G/G A/A 22105026 SNP A G G/G G/G A/A22105286 SNP T C C/C C/C T/T 22105589 SNP A T T/T T/T N/N 22105959 SNP AG G/G G/G A/G 22106046 SNP A G G/G G/G A/A 22106071 SNP T C C/C C/C C/T22106220 SNP T C C/C C/C T/T 22107781 SNP C T C/T C/C C/C 22110491 INS —T T/T T/T —/— 22110813 DEL CAT — —/— —/— CAT/— 22113766 SNP A C C/C C/CA/A 22114123 SNP T A A/A A/A T/T 22114140 SNP A T T/T T/T A/A 22115347SNP A C C/C C/C A/A 22115503 SNP G C C/C C/C G/G 22115913 SNP C T T/TT/T N/N 22117613 SNP C T C/T C/C C/C 22117641 SNP G A A/G G/G G/G22117879 SNP A G A/G A/G A/A 22118166 INS — AT AT/— AT/— —/— 22118600SNP G A A/G A/G N/N 22118730 SNP C G C/G C/C N/N 22119594 SNP G C C/CC/C C/C 22119724 INS — T T/T T/— T/T 22120389 SNP A T A/T A/T A/A

TABLE 8 Allele frequencies of chromosome 9 sequence variants associatedwith CHD in Caucasians. Minor Allele Frequency SNP ID PositionCaucasians African-Americans rs9632884 22062301 0.48 0.008 rs647560622071850 0.5 0.008 rs10757272 22078260 0.5 0.15 rs10757274 22086055 0.490.21 rs4977574 22088574 0.5 0.08 rs2891168 22088619 0.5 0.08 rs133304222093813 0.49 0.008 rs2383206 22105026 0.49 0.41 rs1333048 22115347 0.490.25 rs1333049 22115503 0.49 0.175

TABLE 9 Association between rs10757274 and rs2383206 and CHD inAfrican-Americans in ARIC and DHS. rs10757274 rs2383206 Controls CasesControls Cases Cohort AA AG GG AA AG GG P AA AG GG AA AG GG P ARIC 1857971 143 187 92 14 0.90 1019 1532 541 114 161 46 0.32 DHS 447 251 32 17566 21 0.64 231 371 129 90 123 48 0.67 Values in the table are numbers ofindividuals. P values were calculated using Chi-square tests. ARIC,Atherosclerosis Risk in Communities Study; DHS, Dallas Heart Study.Cases and controls are defined as indicated in the Methods.

TABLE 10 Risk Position Type rs No. Allele MAF 22062264 SNP rs10757269 G0.478 22062301 SNP rs9632884 C 0.478 22062638 SNP rs9632885 A 22062719SNP rs10757270 G 22071397 SNP rs10116277 T 0.494 22071850 SNP rs6475606T 0.494 22078090 SNP rs10738606 T 22078094 SNP rs10738607 G 0.49422078260 SNP rs10757272 T 22078465 DEL — 22086055 SNP rs10757274 G22088574 SNP rs4977574 G 22088619 SNP rs2891168 G 22089755 INS 22090176SNP rs1556516 C 0.494 22091702 SNP rs6475608 C 0.233 22092165 SNPrs7859727 T 0.500 22093183 SNP rs1537372 T 22093341 SNP rs1537373 G0.494 22093813 SNP rs1333042 G 0.489 22095927 SNP C 22096731 SNPrs1333043 A 0.478 22100131 SNP rs1412834 C 0.472 22101587 INS TTGAT22102241 SNP rs7341786 C 0.467 22102427 SNP rs7341791 G 22104469 SNPrs10733376 C 0.478 22104495 SNP rs10738609 G 0.472 22105026 SNPrs2383206 G 0.472 22105286 SNP rs944797 C 0.472 22106046 SNP rs1537374 G0.472 22106220 SNP rs1537376 C 22110491 INS T 22113766 SNP rs10738610 C0.478 22114123 SNP rs1333046 A 0.478 22114140 SNP rs7857118 T 22115347SNP rs1333048 C 0.483 22115503 SNP rs1333049 C 0.500 MAF, minor allelefrequency in the European (CEU) HapMap panel.

TABLE 11 POSITION rs No. MAF 22062730 rs17761197 0.000 22063170rs10965226 0.000 22063334 rs16923583 0.000 22064793 rs7855162 0.00022066071 rs1831733 22066208 rs1831734 22066795 rs10757271 22067085rs10811652 22068305 rs7855660 0.000 22069020 rs6475605 0.000 22070363rs16905613 0.006 22070791 rs7858034 0.000 22071128 rs12347950 0.00022071346 rs1412833 22071796 rs10965227 0.262 22072340 rs1547704 0.00022072380 rs10965228 0.097 22073017 rs7853953 0.000 22074633 rs101221920.000 22076840 rs10120722 0.000 22076883 rs16905635 0.000 22078556rs16905640 0.000 22078937 rs13300638 22079014 rs13284693 0.000 22079193rs12235973 0.000 22080301 rs10757273 22080416 rs10965230 22080416rs35573493 22080521 rs9644859 22080521 rs35170871 22080603 rs964486022080603 rs34933796 22080683 rs7019916 22080753 rs7020031 22080811rs7034707 22081731 rs12005039 22081731 rs34597771 22081924 rs786650322081924 rs34555767 22081924 rs11506225 22082097 rs7869527 22082257rs2210538 22082257 rs11506451 22082551 rs7870178 22082670 rs977654622082924 rs11532910 22082924 rs10811654 22082924 rs34184423 22083299rs34168773 22084281 rs7848875 0.000 22084330 rs35537809 22084330rs7388840 22084330 rs4977757 22084796 rs10738608 22085567 rs3586926122086055 rs10757274 22087022 rs16905644 0.000 22087693 rs6475607 0.00622088038 rs7037832 0.000 22088374 rs1333041 22088574 rs4977574 0.49422088619 rs2891168 0.494 22088674 rs10965231 0.000 22088683 rs1178781422089940 rs7856476 0.000 22090726 rs12238050 0.000 22091120 rs109652320.000 22091259 rs13292938 0.000 22091435 rs7028026 22092128 rs101252310.022 22093314 rs10965233 0.000 22093748 rs7022719 0.000 22094450rs4336695 0.000 22095595 rs7872591 0.000 22098069 rs7855190 0.00022098942 rs10217720 22099387 rs10217426 0.000 22100478 rs17761319 0.00022101973 rs16905648 22102606 rs17834367 0.006 22102943 rs7032115 0.00022103324 rs13301964 0.011 22103924 rs16905652 0.000 22105078 rs109652340.000 22105105 rs10965235 0.000 22105217 rs4990722 0.000 22105285rs944796 0.000 22105633 rs10965236 0.000 22107669 rs7851006 22108026rs17834457 0.000 22108102 rs17761446 0.000 22108378 rs7854631 0.00022108481 rs4977758 0.000 22108885 rs4977759 0.000 22109128 rs13330440.000 22109195 rs1333045 0.442 22111167 rs12685422 0.000 22111349rs10217586 22111353 rs7860589 22112193 rs7020671 22112530 rs109652370.000 22112912 rs13285121 22113590 rs7869069 0.000 22113967 rs78540160.000 22114368 rs17761458 0.000 22114450 rs10757277 22114472 rs1081165622114477 rs10757278 0.488 22114504 rs1333047 0.500 22114630 rs1075727922114744 rs4977575 0.500 22116454 rs12345199 0.000 22116885 rs123361060.000 22117777 rs17834529 22117883 rs10965238 0.030 22117965 rs109652390.000 22118180 rs12379111 0.138 22118709 rs12347779 0.089 22119164rs10965240 0.011 22119579 rs7020996 0.194 22120065 rs10965243 0.144 MAF,minor allele frequency in the European (CEU) HapMap panel.

REFERENCES CITED IN THE EXAMPLE

-   1. C. J. Murray, A. D. Lopez, Lancet 349, 1436 (1997).-   2. C. D. Mathers, D. Loncar, PLoS Med 3, e442 (2006).-   3. S. F. Saccone et al., Hum Mol Genet 16, 36 (2007).-   4. The ARIC Study Investigators, Am J Epidemiol 129, 687 (1989)-   5. P. Schnohr, G. Jensen, H. Scharling, M. Appleyard, Eur Heart J 3.    Suppl H., H1 (2001).-   6. R. G. Victor et al., Am J Cardiol 93, 1473 (2004).-   7. A. S. Agatston et al., J Am Coll Cardiol 15, 827 (1990).-   8. E. Pasmant, Laurendeau, I., Heron, D., Vidaud, M., Vidaud, D.,    Bieche, I., Cancer Res 67, 1 (2007).-   9. J. Cohen et al., Proc Natl Acad Sci USA 103, 1810 (2006).-   10. J. C. Cohen, E. Boerwinkle, T. H. Mosley, H. H. Hobbs, N.    Engl. J. Med. 334, 34 (2006).-   11. S. Romeo et al., Nat Genet 39, 513 (2007).-   12. Circulation 106, 3143 (2002).-   13. The ARIC Study Investigators, Am J Epidemiol 129, 687 (1989).-   14. L. E. Chambless et al., J Clin Epidemiol 56, 880 (2003).-   15. S. A. Brown et al., Arteriscler. Thromb 13, 1139 (1993).-   16. P. Schnohr, G. Jensen, H. Scharling, M. Appleyard, Eur Heart J    3, Suppl H., H1 (2001).-   17. T. Jain et al., J. Am Coll Cardiol 44, 1011 (2004, 2004).-   18. N. Patil et al., Science 294, 1719 (2001).-   19. M. Tartaglia et al., Nat Genet 39, 75 (2007).

EQUIVALENTS

The examples and detailed description herein are offered by way ofillustration and not by way of limitation. Although the foregoinginvention has been described in some detail by way of illustration andexample for purposes of clarity of understanding, it will be readilyapparent to those of ordinary skill in the art in light of the teachingsof this invention that certain changes and modifications may be madethereto without departing from the spirit or scope of the appendedclaims. Those skilled in the art will recognize, or be able to ascertainusing no more than routine experimentation, many equivalents to thespecific embodiments of the invention described herein. Such equivalentsare intended to be encompassed by the following claims.

All publications, patents and patent applications mentioned in thisspecification are herein incorporated by reference into thespecification to the same extent as if each individual publication,patent or patent application was specifically and individually indicatedto be incorporated herein by reference.

Citation or discussion of a reference herein shall not be construed asan admission that such is prior art to the present invention.

1-30. (canceled)
 31. A method for identifying a human subject atincreased risk for coronary heart disease, comprising using an in vitroassay to detect the presence in the subject of a genetic markerassociated with increased risk for coronary heart disease in apopulation, and assigning to the subject an increased risk for coronaryheart disease according to the presence of the marker, wherein themarker is a polymorphism with a linkage disequilibrium in the populationof at least r²=0.50 with at least one “G” allele at the polymorphicposition defined by rs10757274 in a human subject.
 32. The method ofclaim 31, wherein: a) the human subject is heterozygous for thepolymorphism, b) the human subject is homozygous for the polymorphism,c) the linkage disequilibrium has an r² of at least 0.8, or d) thelinkage disequilibrium has an r² of at least 0.89.
 33. The method ofclaim 31, wherein the coronary heart disease is a myocardial infarctionor coronary atherosclerosis or premature coronary heart disease withonset before the age of 60 years.
 34. The method of claim 31, whereinthe polymorphism is detected using mass spectroscopy, oligonucleotidemicroarray analysis, allele-specific hybridization, allele-specific PCRor sequencing.
 35. The method of claim 31, wherein the polymorphismcomprises at least one of the following: a cytosine at rs9632884, athymine at rs6475606, a thymine at rs10757272, a guanine at rs4977574, aguanine at rs2891168, a guanine at rs1333042, a cytosine at rs1333048,or a cytosine at rs1333049.
 36. The method of claim 31, wherein thepolymorphism comprises at least one of the following positions on humanchromosome 9 as defined by NCBI Build 36.1 Coordinates: a guanine atposition 22062264; a cytosine at position 22062301; an adenine atposition 22062638; a guanine at position 22062719; a thymine at position22071397; a thymine at position 22071850; a thymine at position22078090; a guanine at position 22078094; a thymine at position22078260; a deletion at position 22078465; a guanine at position22088574; a guanine at position 22088619; an insertion at position22089755; a cytosine at position 22090176; a cytosine at position22091702; a thymine at position 22092165; a thymine at position22093183; a guanine at position 22093341; a guanine at position22093813; a cytosine at position 22095927; an adenine at position22096731; a cytosine at position 22100131; an insertion at position22101587; a cytosine at position 22102241; a guanine at position22102427; a cytosine at position 22104469; a guanine at position22104495; a cytosine at position 22105286; a guanine at position22106046; a cytosine at position 22106220; an insertion at position22110491; a cytosine at position 22113766; an adenine at position22114123; a thymine at position 22114140; a cytosine at position22115347; or a cytosine at position
 22115503. 37. A method for detectingsusceptibility for coronary heart disease in a human, comprisingdetecting in a nucleic acid sample isolated from the human the presenceof a genetic marker associated with increased risk for coronary heartdisease in a population, and assigning to the subject an increased riskfor coronary heart disease according to the presence of the marker,wherein the marker is at least one “G” allele at the polymorphicposition defined by rs10757274, or a single nucleotide polymorphism inlinkage disequilibrium in the population of at least r²=0.50 with saidallele.
 38. The method of claim 37, wherein: a) the human subject isheterozygous for the polymorphism, b) the human subject is homozygousfor the polymorphism, c) the linkage disequilibrium has an r² of atleast 0.8, or d) the linkage disequilibrium has an r² of at least 0.89.39. The method of claim 31, wherein the coronary heart disease is amyocardial infarction or coronary atherosclerosis or premature coronaryheart disease with onset before the age of 60 years.
 40. The method ofclaim 37, wherein the allele is detected using mass spectroscopy,oligonucleotide microarray analysis, allele-specific hybridization,allele-specific PCR or sequencing.
 41. The method of claim 37, whereinthe polymorphism comprises at least one of the following: a cytosine atrs9632884, a thymine at rs6475606, a thymine at rs10757272, a guanine atrs4977574, a guanine at rs2891168, a guanine at rs1333042, a cytosine atrs1333048, or a cytosine at rs1333049.
 42. The method of claim 37,wherein the polymorphism comprises at least one of the followingpositions on human chromosome 9 as defined by NCBI Build 36.1Coordinates: a guanine at position 22062264; a cytosine at position22062301; an adenine at position 22062638; a guanine at position22062719; a thymine at position 22071397; a thymine at position22071850; a thymine at position 22078090; a guanine at position22078094; a thymine at position 22078260; a deletion at position22078465; a guanine at position 22088574; a guanine at position22088619; an insertion at position 22089755; a cytosine at position22090176; a cytosine at position 22091702; a thymine at position22092165; a thymine at position 22093183; a guanine at position22093341; a guanine at position 22093813; a cytosine at position22095927; an adenine at position 22096731; a cytosine at position22100131; an insertion at position 22101587; a cytosine at position22102241; a guanine at position 22102427; a cytosine at position22104469; a guanine at position 22104495; a cytosine at position22105286; a guanine at position 22106046; a cytosine at position22106220; an insertion at position 22110491; a cytosine at position22113766; an adenine at position 22114123; a thymine at position22114140; a cytosine at position 22115347; or a cytosine at position22115503.
 43. A method of genotyping a nucleic acid sample isolated froma human diagnosed as at risk for coronary heart disease, or diagnosedwith coronary heart disease, comprising determining in the sample thepresence or absence of a genetic marker associated with increased riskfor coronary heart disease in a population, wherein the marker is atleast one “G” allele at the polymorphic position defined by rs10757274,or a single nucleotide polymorphism that is in linkage disequilibrium inthe population with said allele, wherein the linkage disequilibrium hasan r² of at least 0.50.
 44. The method of claim 43, wherein: a) thehuman subject is heterozygous for the polymorphism, b) the human subjectis homozygous for the polymorphism, c) the linkage disequilibrium has anr² of at least 0.8, or d) the linkage disequilibrium has an r² of atleast 0.89.
 45. The method of claim 43, wherein the coronary heartdisease is a myocardial infarction or coronary atherosclerosis orpremature coronary heart disease with onset before the age of 60 years.46. The method of claim 43, wherein the genotyping is performed usingallele-specific hybridization, allele-specific primer extension,allele-specific PCR, sequencing, single-stranded conformationpolymorphisms detection, or molecular beacon assay.
 47. The method ofclaim 43, wherein the polymorphism comprises at least one of thefollowing: a cytosine at rs9632884, a thymine at rs6475606, a thymine atrs10757272, a guanine at rs4977574, a guanine at rs2891168, a guanine atrs1333042, a cytosine at rs1333048, or a cytosine at rs1333049.
 48. Themethod of claim 43, wherein the polymorphism comprises at least one ofthe following positions on human chromosome 9 as defined by NCBI Build36.1 Coordinates: a guanine at position 22062264; a cytosine at position22062301; an adenine at position 22062638; a guanine at position22062719; a thymine at position 22071397; a thymine at position22071850; a thymine at position 22078090; a guanine at position22078094; a thymine at position 22078260; a deletion at position22078465; a guanine at position 22088574; a guanine at position22088619; an insertion at position 22089755; a cytosine at position22090176; a cytosine at position 22091702; a thymine at position22092165; a thymine at position 22093183; a guanine at position22093341; a guanine at position 22093813; a cytosine at position22095927; an adenine at position 22096731; a cytosine at position22100131; an insertion at position 22101587; a cytosine at position22102241; a guanine at position 22102427; a cytosine at position22104469; a guanine at position 22104495; a cytosine at position22105286; a guanine at position 22106046; a cytosine at position22106220; an insertion at position 22110491; a cytosine at position22113766; an adenine at position 22114123; a thymine at position22114140; a cytosine at position 22115347; or a cytosine at position22115503.
 49. A kit adapted to practice the method of claim 31 andcomprising a probe for said marker.
 50. A kit adapted to practice themethod of claim 37 and comprising a probe for said marker.